Immunogenic compositions and uses thereof

ABSTRACT

This invention generally relates to immunogenic compositions that comprise an RNA component and a polypeptide component. Immunogenic compositions that deliver antigenic epitopes in two different forms—a first epitope from a pathogen, in RNA-coded form; and a second epitope from the same pathogen, in polypeptide form—are effective in inducing immune response to the pathogen. The invention also relates to a kit comprising an RNA-based priming composition and a polypeptide-based boosting composition. The kit may be used for sequential administration of the priming and the boosting compositions.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/130,869 filed Apr. 15, 2014, which is the U.S. National Phase ofInternational Application No. PCT/US2012/045854 field Jul. 6, 2012,which claims the benefit of U.S. Provisional Application No. 61/505,105filed on Jul. 6, 2011; the entire contents of the foregoing applicationsare incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 5, 2012, isnamed PAT54514.txt and is 92,631 bytes in size.

BACKGROUND OF THE INVENTION

Nucleic acid based vaccines are an attractive approach to vaccination.For example, intramuscular (IM) immunization of plasmid DNA encoding anantigen can induce cellular and humoral immune responses and protectagainst challenge. DNA vaccines offer certain advantages overtraditional vaccines using protein antigens, or attenuated pathogens.For example, as compared to protein vaccines, DNA vaccines can be moreeffective in producing a properly folded antigen in its nativeconformation, and in generating a cellular immune response. DNA vaccinesalso do not have some of the safety problems associated with killed orattenuated pathogens. For example, a killed viral preparation maycontain residual live viruses, and an attenuated virus may mutate andrevert to a pathogenic phenotype. DNA vaccines are generally effectivein generating cell mediated immunity (such as interferon-γ secretingantigen-specific T-cells and antigen-specific cytotoxic T-cells), butless effective in generating antibodies against the encoded andexpressed antigen.

WO 99/30733 discloses a method of enhancing immune response to a nucleicacid vaccine by simultaneous administration of the protein that isencoded by the nucleic acid. The two components do not need to beadministered in the same composition. Both components need to beadministered during the induction phase of the immune response, with theprotein preferably being masked or held back until after the nucleicacid has primed the immune system. In some examples, the vaccinecomprised naked DNA and naked protein antigen in physical admixture. Inothers examples, the protein antigen was formulated for delayed releasein a biodegradable polymer-alum formulation admixed with naked DNA.

WO 97/28818 discloses a vaccine that delivers a nucleic acid and aprotein antigen to antigen presenting cells. The nucleic acid may encodethe same protein as the protein antigen. The nucleic acid and proteinare “complexed,” e.g., by covalent conjugation. The complex may beformulated as a synthetic virus-like particle. It is also suggested thatliposomal systems may be used.

U.S. Pat. No. 7,604,803 discloses the co-delivery of nucleic acid andits encoded protein to the same cell using a liposomal system. The DNAmolecule and its encoded protein are entrapped within the same liposomalvehicle, such that the two entities arrive at antigen-presenting cellstogether, resulting in the processing and presentation of the proteinform of the antigen, together with the expression of the DNA-encodedform of the antigen in the same cell.

WO 2009/156852 discloses a prime/boost approach to raise an immuneresponse in a subject. The method comprises: (i) administering at leastone dose of a priming immunogenic composition to the subject, to elicita primary immune response against a pathogen; and (ii) administering aboosting immunogenic composition to the subject, to elicit, within 21days of its administration or sooner, a protective anamnestic immuneresponse against the pathogen.

WO 2009/074861 discloses a vaccine comprising (i) a nucleic acidsequence encoding at least one influenza virus antigen coated ontocarrier particles, and (ii) an assistor protein for sequential orconcomitant administration. The assistor protein and the antigen encodedby the nucleic acid molecule share at least one common epitope.

WO 2006/061643 discloses a prime-boost vaccination method using herpesviral vectors, in particular, a heterologous prime-boost regimen usingtwo different non-replicating viral vectors, one of which is a herpesvirus vector. The two vectors share a common epitope that is associatedwith a target antigen or disease.

WO 2010/036948 discloses the use of a priming composition and a boostingcomposition to prime and boost an immune response. The primingcomposition comprises a DNA plasmid that comprises a nucleic acidmolecule encoding an influenza virus hemagglutinin (HA) or anepitope-bearing domain thereof. The boosting composition comprises aninfluenza vaccine.

It is known that non-coding plasmid DNA has an immuno-adjuvant actionwhen co-entrapped with peptides in liposomal vesicles (Gursel, M. et al.Vaccine (1999) 17: 1376-1383) and that DNA with CpG motifs has anadjuvant effect on naked DNA and peptide vaccines (Klinman, D. M. et al.Vaccine (1999) 17: 19-25).

Concerns have been raised regarding the safety of DNA-based vaccines.The introduced DNA molecules could potentially integrate into the hostgenome or, due to their distribution to various tissues, could lead toundesirable sustained expression of antigens. In addition, certain DNAviruses have also been used to deliver DNA molecules. Because of theirinfectious properties, such viruses achieve a very high transfectionrate. The viruses used are genetically modified to prevent the formationof functional infectious particles in the transfected cell. Despitethese precautions, however, it is not possible to rule out the risk ofuncontrolled propagation of the introduced gene and viral genes, forexample due to potential recombination events. This also entails therisk of the DNA being inserted into an intact gene of the host cell'sgenome by e.g. recombination, with the consequence that the host genemay be mutated and thus completely or partially inactivated or may giverise to misinformation. In other words, synthesis of a host gene productwhich is vital to the cell may be completely suppressed or,alternatively, a modified or incorrect gene product is expressed.

RNA molecules encoding an antigen or a derivative thereof may also beused as vaccines. RNA vaccines offer certain advantages as compared toDNA vaccines. However, compared with DNA-based vaccines, relativelyminor attention has been given to RNA-based vaccines. RNAs are highlysusceptible to degradation by nucleases when administered as atherapeutic or vaccine. Additionally, RNAs are not actively transportedinto cells. See, e.g., Vajdy, M., et al., Mucosal adjuvants and deliverysystems for protein-, DNA- and RNA-based vaccines, Immunol Cell Biol,2004. 82(6): p. 617-27.

Toll-like receptors (TLRs) are a group of pattern recognition receptorswhich bind to pathogen-associated molecular patterns (PAMPS) frombacteria, fungi, protozoa and viruses, and act as a first line ofdefense against invading pathogens. Many TLRs have been identified inhumans, mice, and other mammalian species. DNA molecules (such asbacterial or viral DNA) are recognized by TLR9, whereas RNA molecules(such as single stranded viral RNA) are recognized by TLR7 or TLR8.

U.S. Pat. No. 7,862,829 discloses a method of producing an immuneresponse by administering an antigen and an alphavirus-based adjuvant.The method is based on the discovery that alphavirus, a (+)ssRNA virus,can act as an adjuvant to enhance an immune response against an antigen,even though the antigen is not presented on or expressed by the virus.The alphavirus particles may be delivered by liposomal system.

There is a need to improve the efficacy of protein subunit vaccines andnucleic acid vaccines such as RNA vaccines.

SUMMARY OF THE INVENTION

Certain terms that are used to describe the invention in this aredefined and explained herein in Section 6.

This invention generally relates to immunogenic compositions thatcomprise an RNA component and a polypeptide component. Immunogeniccompositions that deliver antigenic epitopes in two different forms—afirst epitope from a pathogen, in RNA-coded form; and a second epitopefrom the same pathogen, in polypeptide form—can enhance the immuneresponse to the pathogen, as compared to immunization with RNA alone, orpolypeptide alone. Preferably, the first epitope and the second epitopeare the same epitope.

The invention also relates to a kit comprising an RNA-based primingcomposition and a polypeptide-based boosting composition for sequentialadministration. The kit is suitable for, for example, a “RNA prime,protein boost” immunization regimen to generate an immune response to apathogen.

The invention also relates to methods for treating or preventing aninfectious disease, methods for inducing an immune response, or methodsof vaccinating a subject, by co-delivery of an RNA molecule and apolypeptide molecule (co-administration).

The invention also relates to methods for treating or preventing aninfectious disease, methods for inducing an immune response, or methodsof vaccinating a subject, by sequential administration of an RNAmolecule and a polypeptide molecule (prime-boost).

In one aspect, the invention provides an immunogenic compositioncomprising: (i) a self-replicating RNA molecule that encodes a firstpolypeptide antigen comprising a first epitope; and (ii) a secondpolypeptide antigen comprising a second epitope; wherein the first andsecond epitope are epitopes from the same pathogen. The first and andsecond epitopes can be the same epitope. The first and second epitopescan be different epitopes.

In some embodiments, the first polypeptide antigen and the secondpolypeptide antigen are substantially the same.

The first polypeptide antigen can be a soluble or membrane anchoredpolypeptide and the second polypeptide antigen can be a solublepolypeptide.

In some embodiments, the first polypeptide antigen is a fusionpolypeptide that further comprises a third epitope from a differentpathogen. In some embodiments, the second polypeptide antigen is afusion polypeptide that further comprises a third epitope from adifferent pathogen. The first and second epitopes can be epitopes fromthe same subspecies of the pathogen.

The self-replicating RNA can be an alphavirus-derived RNA replicon. Theself-replicating RNA molecule can comprise one or more modifiednucleotides.

In some embodiments, the immunogenic composition further comprises acationic lipid, a liposome, a cochleate, a virosome, animmune-stimulating complex, a microparticle, a microsphere, ananosphere, a unilamellar vesicle, a multilamellar vesicle, anoil-in-water emulsion, a water-in-oil emulsion, an emulsome, apolycationic peptide, or a cationic nanoemulsion.

In some embodiments, the RNA molecule is encapsulated in, bound to oradsorbed on a cationic lipid, a liposome, a cochleate, a virosome, animmune-stimulating complex, a microparticle, a microsphere, ananosphere, a unilamellar vesicle, a multilamellar vesicle, anoil-in-water emulsion, a water-in-oil emulsion, an emulsome, apolycationic peptide, a cationic nanoemulsion or combinations thereof.

In some embodiments, the pathogen is a virus, and the first polypeptideantigen and second polypeptide antigen are viral antigens. The viralantigens can be RSV-F antigens. Preferably, the RSV-F antigens comprisean amino acid sequence selected from SEQ ID NOs:25-40.

The viral antigens can be from Cytomegalovirus (CMV). In someembodiments, the CMV antigens are independently selected from the groupconsisting of a gB antigen, a gH antigen, a gL antigen, a gM antigen, agN antigen, a gO antigen, a UL128 antigen, a UL129 antigen, and a UL130antigen.

In some embodiments, the immunogenic composition further comprises anadjuvant. The adjuvant can be MF59.

The invention also relates to pharmaceutical compositions that comprisean immunogenic composition as described herein and a pharmaceuticallyacceptable carrier and/or a pharmaceutically acceptable vehicle.

The invention also relates to methods for treating or preventing aninfectious disease comprising administering to a subject in need thereofa therapeutically effective amount of a composition as described herein.

The invention also relates to methods of inducing an immune response ina subject comprising administering to a subject in need thereof atherapeutically effective amount of a composition as described herein.

The invention also relates to methods of vaccinating a subject,comprising administering to a subject in need thereof a composition asdescribed herein.

The invention also relates to kits comprising: (i) a priming compositioncomprising a self-replicating RNA molecule that encodes a firstpolypeptide antigen that comprises a first epitope from a pathogen; and(ii) a boosting composition comprising a second polypeptide antigen thatcomprises a second epitope from the pathogen. The first and secondpolypeptide antigens can be substantially the same. The firstpolypeptide antigen can be a soluble or membrane anchored polypeptide,and the second polypeptide antigen can be a soluble polypeptide. Thefirst polypeptide antigen can be a fusion polypeptide. The secondpolypeptide antigen can be a fusion polypeptide. The self-replicatingRNA can be an alphavirus-derived RNA replicon. The self-replicating RNAmolecule can comprise one or more modified nucleotides.

In some embodiments, the priming composition of the kit furthercomprises a cationic lipid, a liposome, a cochleate, a virosome, animmune-stimulating complex, a microparticle, a microsphere, ananosphere, a unilamellar vesicle, a multilamellar vesicle, anoil-in-water emulsion, a water-in-oil emulsion, an emulsome, apolycationic peptide, or a cationic nanoemulsion.

In some embodiments, the RNA molecule of the kit is encapsulated in,bound to or adsorbed on a cationic lipid, a liposome, a cochleate, avirosome, an immune-stimulating complex, a microparticle, a microsphere,a nanosphere, a unilamellar vesicle, a multilamellar vesicle, anoil-in-water emulsion, a water-in-oil emulsion, an emulsome, apolycationic peptide, a cationic nanoemulsion or combinations thereof.

In some embodiments, the pathogen of the kit is a virus, and the firstpolypeptide antigen and second polypeptide antigen are viral antigens.The viral antigens can be from respiratory syncytial virus (RSV). Theviral antigens can be a RSV-F antigen. Preferably, the RSV-F antigenscomprise an amino acid sequence selected from SEQ ID NOs:25-40.

In some embodiments, the viral antigens are from Cytomegalovirus (CMV).In some embodiments, the CMV antigens are independently selected fromthe group consisting of a gB antigen, a gH antigen, a gL antigen, a gMantigen, a gN antigen, a gO antigen, a UL128 antigen, a UL129 antigen,and a UL130 antigen.

In some embodiments, the priming composition of the kit, the boostingcomposition of the kit, or both, comprise an adjuvant. The adjuvant canbe MF59. The priming composition, the boosting composition, or both cancomprise a pharmaceutically acceptable carrier and/or a pharmaceuticallyacceptable vehicle.

The invention also relates to methods for treating or preventing aninfectious disease comprising: (i) administering to a subject in needthereof at least once a therapeutically effective amount of a primingcomposition comprising a self-replicating RNA molecule that encodes afirst polypeptide antigen that comprises a first epitope from apathogen; and subsequently administering the subject at least once atherapeutically effective amount of a boosting composition comprising asecond polypeptide antigen that comprises a second epitope from saidpathogen; wherein said first epitope and second epitope are the sameepitope.

The invention also relates to methods for inducing an immune response ina subject comprising (i) administering to a subject in need thereof atleast once a therapeutically effective amount of a priming compositioncomprising a self-replicating RNA molecule that encodes a firstpolypeptide antigen that comprises a first epitope from a pathogen; andsubsequently administering the subject at least once a therapeuticallyeffective amount of a boosting composition comprising a secondpolypeptide antigen that comprises a second epitope from said pathogen;wherein said first epitope and second epitope are the same epitope.

The invention also relates to methods for vaccinating a subject,comprising administering to a subject in need thereof at least once atherapeutically effective amount of a priming composition comprising aself-replicating RNA molecule that encodes a first polypeptide antigenthat comprises a first epitope from a pathogen; and subsequentlyadministering the subject at least once a therapeutically effectiveamount of a boosting composition comprising a second polypeptide antigenthat comprises a second epitope from said pathogen; wherein said firstepitope and second epitope are the same epitope.

In some embodiments, the first polypeptide antigen and the secondpolypeptide antigen are substantially the same.

The first polypeptide antigen can be a soluble or membrane anchoredpolypeptide and the second polypeptide antigen can be a solublepolypeptide.

In some embodiments, the first polypeptide antigen is a fusionpolypeptide that further comprises a third epitope from a differentpathogen. In some embodiments, the second polypeptide antigen is afusion polypeptide that further comprises a third epitope from adifferent pathogen. The first and second epitopes can be epitopes fromthe same subspecies of the pathogen.

The self-replicating RNA can be an alphavirus-derived RNA replicon. Theself-replicating RNA molecule can comprise one or more modifiednucleotides.

In some embodiments, the immunogenic composition further comprises acationic lipid, a liposome, a cochleate, a virosome, animmune-stimulating complex, a microparticle, a microsphere, ananosphere, a unilamellar vesicle, a multilamellar vesicle, anoil-in-water emulsion, a water-in-oil emulsion, an emulsome, apolycationic peptide, or a cationic nanoemulsion.

In some embodiments, the RNA molecule is encapsulated in, bound to oradsorbed on a cationic lipid, a liposome, a cochleate, a virosome, animmune-stimulating complex, a microparticle, a microsphere, ananosphere, a unilamellar vesicle, a multilamellar vesicle, anoil-in-water emulsion, a water-in-oil emulsion, an emulsome, apolycationic peptide, a cationic nanoemulsion or combinations thereof.

In some embodiments, the pathogen is a virus, and the first polypeptideantigen and second polypeptide antigen are viral antigens. The viralantigens can be RSV-F antigens. Preferably, the RSV-F antigens comprisean amino acid sequence selected from SEQ ID NOs:25-40.

The viral antigens can be from Cytomegalovirus (CMV). In someembodiments, the CMV antigens are independently selected from the groupconsisting of a gB antigen, a gH antigen, a gL antigen, a gM antigen, agN antigen, a gO antigen, a UL128 antigen, a UL129 antigen, and a UL130antigen.

In some embodiments, the immunogenic composition further comprises anadjuvant. The adjuvant can be MF59.

The invention also relates to pharmaceutical compositions that comprisean immunogenic composition as described herein and a pharmaceuticallyacceptable carrier and/or a pharmaceutically acceptable vehicle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing neutralizing antibody tires elicited by athree dose immunization regimen in which VRPs that express gH/gL, VRPthat express green fluorescent protein (VRP-G), and purified gH/gLadministered that were administered in various orders or were mixed andcoadministered. (see Example VI, Table VI-1) Key: VRP=gH/gL expressingVRP, VRP-G=green fluorescent protein expressing VRP, PBS=gHsol/gLsubunit in PBS, MF59=gHsol/gL in MF59, Mix=gH/gL VRP+gHsol/gL (noadjuvant), Mix-G=VRP-G+gHsol/gL (no adjuvant).

FIG. 2 is a graph showing the percentage of cytokine positiveantigen-specific CD4 T cells at 4wp3 (day 69) after immunization usingthe dose immunization regimen described in FIG. 1 and Example VI, TableVI-1. Splenocytes were restimulated in vitro with gHsol/gL protein.

FIG. 3 is a graph showing the percentage of cytokine positiveantigen-specific CD8 T cells at 4wp3 (day 69) after immunization usingthe dose immunization regimen described in FIG. 1 and Example VI, TableVI-1. Splenocytes were restimulated in vitro with gH peptide pool 2.

FIG. 4 is a graph showing self amplifying RNA and subunit, alone or incombination, elicit high neutralizing antibody titers. The selfamplifying RNA was encapsulated in LNPs. Self amplifying RNA and subunitdose was 1 μg, mixed dose was 1+1 μg. Neutralizing assay: VR1814infection of ARPE-19 cells in presence of complement.

FIGS. 5A and 5B are graphs showing CD4+ T cell responses to thevaccinations using purified gH/gL and pentameric subunits at (A) 3wp3(day 64) and (B) 4wp3 (day 71).

FIGS. 6A and 6B are graphs showing CD4+ T cell responses to thevaccinations using purified pentameric complex at (A) 3wp3 (day 64) and(B) 4wp3 (day 71).

FIGS. 7A and 7B are graphs showing CD8+ T cell responses to thevaccinations using purified pentameric complex at (A) 3wp3 (day 64) and(B) 4wp3 (day 71).

FIGS. 7A and 7B are graphs showing CD8+ T cell responses to thevaccinations using purified pentameric complex at (A) 3wp3 (day 64) and(B) 4wp3 (day 71).

FIGS. 8A and 8B are graphs showing CD8+ T cell responses to thevaccinations using gH peptide pool 2 at (A) 3wp3 (day 64) and (B) 4wp3(day 71).

FIG. 9 is a graph showing high neutralizing antibody titers in Balb/cmice that were administered RSV-F-RNA vaccine prime, followed by RSV-Fsubunit vaccine boost. Mice were administered the vaccineintramuscularly on days 0, 21 and 44, and bleeds were taken on days 35and 57.

FIG. 10 is a graph showing the F-specific IgG2a:IgG1 ratio in Balb/cmice that were administered the RSV-F-RNA vaccine. The F-RNA vaccine ispotent at setting a Th1 immune response, that is maintained after a Fsubunit vaccine boost. Mice were administered vaccinationsintramuscularly on days 0, 21 and 44, and bleeds were taken on days 35and 57. The spleens of the mice were harvested on day 57.

FIGS. 11A-11C are graphs showing the effect of adjuvant on subunitboost. Even though a very low dose of RNA (300 pg) does not induce ameasurable RSV-specific antibody response in BALB/c mice (FIG. 11A), itis sufficient to set a Th1-biased phenotype after a subunit vaccineboost (IgG2a-biased, FIG. 11B; IFNγ, FIG. 11C; no significant IL-5, FIG.11C). Subunit vaccine adjuvant (none, alum or MF59) has little impact onthe magnitude of the neutralizing antibody titer induced by RNA vaccineprime, subunit vaccine boost.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

One particular advantage of an RNA vaccine is that RNA molecules areself-adjuvanting. For example, the inventors observed that RNA molecules(formulated in liposomes) induced several serum cytokines, includingIFN-α, IP-10 (CXCL-10), IL-6, KC (CXCL1), IL-5, IL-13, MCP-1, and MIP-α,within 24 hours of intramuscular injection into a mouse model. Thecytokines can enhance the host immune response to the protein antigenthat was encoded by the RNA molecule.

Vaccination strategies that combine an RNA molecule and a polypeptidemolecule (e.g., administering an immunogenic composition that has an RNAcomponent and a protein component; or sequential administration regimenssuch as “RNA prime, protein boost”) provide several benefits. Forexample, the polypeptide molecule can enhance total antibody titers inthe host, while the RNA molecule can enhance the production ofantibodies that recognize an antigen in its native structure. Thus thecombination can induce an antibody response with an enhanced ratio offunctional antibodies (e.g., neutralizing antibodies) to totalantibodies. Furthermore, RNA molecules promote type 1 T helper responses(Th1, IFN-γ^(hi), IL-4^(lo)), whereas protein molecules promote type 2 Thelper responses. Thus, combining an RNA molecule and a polypeptidemolecule can promote both T cell-mediated immunity as well as humoralimmunity. In addition, RNA molecules may be delivered to cells usingdelivery systems such as liposomes or oil-in-water emulsions. Liposomesand oil-in-water emulsions are also known to have adjuvant activities.Thus, the adjuvant activity of the RNA together with adjuvant activityof the delivery system can act synergistically to enhance the immuneresponse to an antigen. Finally, multivalency may be achieved bycombining a polypeptide antigen with an RNA that encodes a differentantigen from the same pathogen.

(A) Co-Administration of an RNA Molecule and a Polypeptide Molecule

In one aspect, the invention relates to immunogenic compositions thatcomprise an RNA component and a polypeptide component. Immunogeniccompositions that deliver antigenic epitopes in two different forms—afirst epitope from a pathogen, in RNA-coded form; and a second epitopefrom the same pathogen, in polypeptide form—can enhance the immuneresponse to the pathogen.

Preferably, the first epitope and the second epitope are the sameepitope (i.e., the first antigen, in RNA-coded form, and the secondantigen, in polypeptide form, share at least one common epitope). Forexample, the RNA component of the immunogenic composition can encode aprotein that is substantially the same as the polypeptide component ofthe immunogenic composition (e.g., the amino acid sequence encoded bythe RNA molecule and the polypeptide component of the immunogeniccomposition share at least about 90% sequence identity across the lengthof the shorter antigen). Alternatively, the two antigens have the sameepitope, such as the same immunodominant epitope(s).

As described herein, the inventors have evaluated the efficacies ofimmunogenic compositions that comprise (i) a self-replicating RNAmolecule that encodes a viral antigen, and (ii) the viral antigen inpolypeptide form. Viral antigens that were used in these studies includeHIV gp140 and RSV-F antigens. The results demonstrated thatco-administering an RNA molecule that encodes a viral antigen, togetherwith the viral antigen in polypeptide form, potentiated the immuneresponse to the antigen, resulting in higher antibody titers as comparedto administering the RNA molecule alone. In addition, co-administering aviral antigen in RNA-coded form and in polypeptide form enhanced isotypeswitching from IgG₁ to IgG_(2a), producing a more balanced IgG₁:IgG_(2a)subtype profile as compared to administering the polypeptide antigenalone. Finally, the studies disclosed herein also show thatadministrating an antigen in RNA-coded form and polypeptide from canenhance CD4+ and CD8+ T cell-mediated immunity.

The immunogenic compositions described herein can be formulated as avaccine to induce or enhance the host immune response to a pathogen.Also provided herein are methods of using the immunogenic compositionsof the invention to induce or enhance an immune response in a subject inneed thereof.

(B) Prime-Boost

In another aspect, the invention relates to a kit comprising: (i) apriming composition comprising a self-replicating RNA molecule thatencodes a first polypeptide antigen that comprises a first epitope, and(ii) a boosting composition comprising a second polypeptide antigen thatcomprises a second epitope; wherein said first epitope and secondepitope are the same epitope (i.e., the first antigen, in RNA-codedform, and the second antigen, in polypeptide form, share at least onecommon epitope). The kit may be used for sequential administration ofthe priming and the boosting compositions.

In another aspect, the invention relates to a method for treating orpreventing an infectious disease, a method for inducing an immuneresponse in a subject, or a method of vaccinating a subject, comprising:(i) administering to a subject in need thereof at least once atherapeutically effective amount of a priming composition comprising aself-replicating RNA molecule that encodes a first polypeptide antigenthat comprises a first epitope, and (ii) subsequently administering thesubject at least once a therapeutically effective amount of a boostingcomposition comprising a second polypeptide antigen that comprises asecond epitope; wherein said first epitope and second epitope are thesame epitope (i.e., the first antigen, in RNA-coded form, and the secondantigen, in polypeptide form, share at least one common epitope).

As described herein, the inventors have evaluated RNA prime, proteinboost vaccination strategies. These studies demonstrate several benefitsof the RNA prime, protein boost strategy, as compared to a proteinprime, protein boost strategy, including, for example, increasedantibody titers, a more balanced IgG₁:IgG_(2a) subtype profile,induction of T_(H)1 type, CD4+ T cell-mediated immune response that wassimilar to that of viral particles, and reduced production ofnon-neutralizing antibodies.

Preferably, the RNA molecule in the priming composition encodes aprotein that is substantially the same as the polypeptide molecule inthe boosting composition (e.g., the amino acid sequence encoded by theRNA molecule in the priming composition and the polypeptide in theboosting composition share at least about 90% sequence identity acrossthe length of the shorter antigen). Alternatively, the two antigens havethe same epitope, such as the same immunodominant epitope(s).

The priming and boosting compositions described herein can be formulatedas a vaccine to induce or enhance the immune response to a pathogen.Also provided herein are methods of using the priming and boostingcompositions of the invention to induce or enhance an immune response ina subject in need thereof.

The invention also relates to immunogenic compositions, pharmaceuticalcompositions, or kits as described herein for use in therapy, and to theuse of immunogenic compositions, pharmaceutical compositions, or kits asdescribed herein for the manufacture of a medicament for enhancing orgenerating an immune response.

2. Immunogenic Compositions

In one aspect, the invention provides an immunogenic compositioncomprising an RNA component and a polypeptide component. The immunogeniccomposition comprises: (i) a self-replicating RNA molecule that encodesa first polypeptide antigen comprising a first epitope (the RNAcomponent); and (ii) a second polypeptide antigen comprising a secondepitope (the polypeptide component); wherein said first epitope andsecond epitope are epitopes from the same pathogen.

The first epitope and second epitope can be the same epitope, ordifferent epitopes if desired. The first epitope and second epitope canbe from the same polypeptide of the pathogen, or different polypeptidesof the pathogen. The first epitope and second epitope can also beepitopes which are highly conserved between different strains orsubspecies of the pathogen, such as those epitopes with limited or nomutational variations. For example, the first epitope and the secondepitope can be different epitopes from the same pathogen (e.g., thefirst epitope is from RSV F and the second epitope is from RSV G).

In certain embodiments, the first polypeptide antigen and the secondpolypeptide antigen are derived from the same protein from the pathogen.For example, the RNA molecule may encode a first polypeptide antigencomprising a full-length protein from a pathogen (e.g., a viralprotein), or an antigenic portion thereof, optionally fused with aheterologous sequence that may facilitate the expression, production,purification or detection of the viral protein encoded by the RNA. Thesecond polypeptide antigen may be a recombinant protein comprising thefull-length protein, or an antigenic portion thereof, optionally fusedwith a heterologous sequence (e.g., His-tag) that may facilitate theexpression, production, purification or detection of the secondpolypeptide antigen. Alternatively, the first polypeptide antigen, thesecond polypeptide antigen, or both, may comprise a mutation variant ofa protein from a pathogen (e.g., a viral protein having amino acidsubstitution(s), addition(s), or deletion(s)).

Preferably, the amino acid sequence identity between the firstpolypeptide antigen and the second polypeptide antigen is at least about40%, least about 50%, least about 60%, least about 65%, least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%. In certain embodiments, thefirst polypeptide antigen and the second polypeptide antigen are thesame antigen.

In certain embodiments, the first polypeptide antigen and thepolypeptide second antigen share at least 1, at least 2, at least 3, atleast 4, or at least 5 common B-cell or T-cell epitopes. In certainembodiments, the first polypeptide antigen and the second polypeptideantigen have at least one common immunodominant epitope. In certainembodiments, the first polypeptide antigen and the second polypeptideantigen have the same immunodominant epitope(s), or the same primaryimmunodominant epitope.

In certain embodiments, the first polypeptide antigen is a soluble ormembrane anchored polypeptide, and the second polypeptide antigen is asoluble polypeptide. For example, if the wild type viral protein is atransmembrane surface protein, the RNA molecule may comprise thefull-length coding sequence to produce the first (membrane-anchored)antigen, while the transmembrane region of the viral protein may bedeleted to produce the second polypeptide antigen (which is soluble).

In certain embodiment, the first antigen or the second antigen is afusion polypeptide further comprising a third epitope. The third epitopemay be from a different pathogen, or from a different antigen of thesame pathogen.

A. Antigens

Antigens suitable for inclusion in the immunogenic compositionsdescribed herein (either in RNA-coded form or in polypeptide form) maybe derived from any pathogen (e.g., a bacterial pathogen, a viralpathogen, a fungal pathogen, a protozoan pathogen, or a multi-cellularparasitic pathogen), allergen or tumor.

In certain embodiments, the first and second antigens are derived from aviral pathogen. Exemplary viral pathogens include, e.g., respiratorysyncytial virus (RSV), hepatitis B virus (HBV), hepatitis C virus (HCV),Dengue virus, herpes simplex virus (HSV; e.g., HSV-I, HSV-II), molluscumcontagiosum virus, vaccinia virus, variola virus, lentivirus, humanimmunodeficiency virus (HIV), human papilloma virus (HPV),cytomegalovirus (CMV), varicella zoster virus (VZV), rhinovirus,enterovirus, adenovirus, coronavirus (e.g., SARS), influenza virus(flu), para-influenza virus, mumps virus, measles virus, papovavirus,hepadnavirus, flavivirus, retrovirus, arenavirus (e.g., LymphocyticChoriomeningitis Virus, Junin virus, Machupo virus, Guanarito virus, orLassa virus), norovirus, yellow fever virus, rabies virus, filovirus(e.g., Ebola virus or marbug virus), hepatitis C virus, hepatitis Bvirus, hepatitis A virus, Morbilliviruses (e.g., measles virus),Rubulaviruses (e.g., mumps virus), Rubiviruses (e.g., rubella virus),bovine viral diarrhea virus. For example, the antigen can be CMVglycoprotein gH, or gL; Parvovirus; HIV glycoprotein gp120 or gp140, HIVp55 gag, pol; or RSV-F antigen, etc.

In some embodiments, the first and second antigens are derived from avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of atlantic salmon), landlocked salmon virus (LSV), atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

In some embodiments the first and second antigens are derived from aparasite from the Plasmodium genus, such as P. falciparum, P. vivax, P.malariae or P. ovale. Thus the invention may be used for immunisingagainst malaria. In some embodiments the first and second antigens arederived from a parasite from the Caligidae family, particularly thosefrom the Lepeophtheirus and Caligus genera e.g. sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

In certain embodiments, first and second antigens are derived from abacterial pathogen. Exemplary bacterial pathogens include, e.g.,Neisseria spp, including N. gonorrhea and N. meningitides; Streptococcusspp, including S. pneumoniae, S. pyogenes, S. agalactiae, S. mutans;Haemophilus spp, including H. influenzae type B, non typeable H.influenzae, H. ducreyi; Moraxella spp, including M. catarrhalis, alsoknown as Branhamella catarrhalis; Bordetella spp, including B.pertussis, B. parapertussis and B. bronchiseptica; Mycobacterium spp.,including M. tuberculosis, M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli,enterohemorragic E. coli, enteropathogenic E. coli; Vibrio spp,including V. cholera, Shigella spp, including S. sonnei, S. dysenteriae,S. flexnerii; Yersinia spp, including Y. enterocolitica, Y. pestis, Y.pseudotuberculosis, Campylobacter spp, including C. jejuni and C. coli;Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S.enteritidis; Listeria spp., including L. monocytogenes; Helicobacterspp, including H pylori; Pseudomonas spp, including P. aeruginosa,Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcusspp., including E. faecalis, E. faecium; Clostridium spp., including C.tetani, C. botulinum, C. difficile; Bacillus spp., including B.anthracis; Corynebacterium spp., including C. diphtheriae; Borreliaspp., including B. burgdorferi, B. garinii, B. afzelii, B. andersonii,B. hermsii; Ehrlichia spp., including E. equi and the agent of the HumanGranulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii;Chlamydia spp., including C. trachomatis, C. neumoniae, C. psittaci;Leptsira spp., including L. interrogans; Treponema spp., including T.pallidum, T. denticola, T. hyodysenteriae.

In certain embodiments, first and second antigens are derived from afungal pathogen (e.g., a yeast or mold pathogen). Exemplary fungalpathogens include, e.g., Aspergillus fumigatus, A. flavus, A. niger, A.terreus, A. nidulans, Coccidioides immitis, Coccidioides posadasii,Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, andPneumocystis jirovecii.

In certain embodiments, first and second antigens are derived from aprotozoan pathogen. Exemplary protozoan pathogens include, e.g.,Toxoplasma gondii and Strongyloides stercoralis.

In certain embodiments, the first and second antigens are derived from amulti-cellular parasitic pathogen. Exemplary multicellular parasiticpathogens include, e.g., trematodes (flukes), cestodes (tapeworms),nematodes (roundworms), and arthropods.

In some embodiments, the first and second antigens are derived from anallergen, such as pollen allergens (tree-, herb, weed-, and grass pollenallergens); insect or arachnid allergens (inhalant, saliva and venomallergens, e.g. mite allergens, cockroach and midges allergens,hymenopthera venom allergens); animal hair and dandruff allergens (frome.g. dog, cat, horse, rat, mouse, etc.); and food allergens (e.g. agliadin). Important pollen allergens from trees, grasses and herbs aresuch originating from the taxonomic orders of Fagales, Oleales, Pinalesand platanaceae including, but not limited to, birch (Betula), alder(Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar(Cryptomeria and Juniperus), plane tree (Platanus), the order of Poalesincluding grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis,Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales andUrticales including herbs of the genera Ambrosia, Artemisia, andParietaria. Other important inhalation allergens are those from housedust mites of the genus Dermatophagoides and Euroglyphus, storage mitee.g. Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches,midges and fleas e.g. Blatella, Periplaneta, Chironomus andCtenocepphalides, and those from mammals such as cat, dog and horse,venom allergens including such originating from stinging or bitinginsects such as those from the taxonomic order of Hymenoptera includingbees (Apidae), wasps (Vespidea), and ants (Formicoidae).

In some embodiments, the first and second antigens are derived from atumor antigen selected from: (a) cancer-testis antigens such asNY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE familypolypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3,MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, toaddress melanoma, lung, head and neck, NSCLC, breast, gastrointestinal,and bladder tumors; (b) mutated antigens, for example, p53 (associatedwith various solid tumors, e.g., colorectal, lung, head and neckcancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer andcolorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1(associated with, e.g., melanoma), caspase-8 (associated with, e.g.,head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer),HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR(associated with, e.g., T-cell non-Hodgkins lymphoma), BCR-abl(associated with, e.g., chronic myelogenous leukemia), triosephosphateisomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) over-expressed antigens,for example, Galectin 4 (associated with, e.g., colorectal cancer),Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3(associated with, e.g., chronic myelogenous leukemia), WT 1 (associatedwith, e.g., various leukemias), carbonic anhydrase (associated with,e.g., renal cancer), aldolase A (associated with, e.g., lung cancer),PRAME (associated with, e.g., melanoma), HER-2/neu (associated with,e.g., breast, colon, lung and ovarian cancer), mammaglobin,alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associatedwith, e.g., colorectal cancer), gastrin (associated with, e.g.,pancreatic and gastric cancer), telomerase catalytic protein, MUC-1(associated with, e.g., breast and ovarian cancer), G-250 (associatedwith, e.g., renal cell carcinoma), p53 (associated with, e.g., breast,colon cancer), and carcinoembryonic antigen (associated with, e.g.,breast cancer, lung cancer, and cancers of the gastrointestinal tractsuch as colorectal cancer); (d) shared antigens, for example,melanoma-melanocyte differentiation antigens such as MART-1/Melan A,gp100, MC1R, melanocyte-stimulating hormone receptor, tyrosinase,tyrosinase related protein-1/TRP1 and tyrosinase related protein-2/TRP2(associated with, e.g., melanoma); (e) prostate associated antigens suchas PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g.,prostate cancer; (f) immunoglobulin idiotypes (associated with myelomaand B cell lymphomas, for example). In certain embodiments, tumorimmunogens include, but are not limited to, p15, Hom/Mel-40, H-Ras,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA,human papillomavirus (HPV) antigens, including E6 and E7, hepatitis Band C virus antigens, human T-cell lymphotropic virus antigens, TSP-180,p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72,beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242,CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein/cyclophilin C-associated protein), TAAL6, TAG72, TLP,TPS, and the like.

1. RSV

In some aspects, the pathogen is RSV. RSV is an enveloped non-segmentednegative-strand RNA virus in the family Paramyxoviridae, genusPneumovirus. To infect a host cell, paramyxoviruses such as RSV, likeother enveloped viruses such as influenza virus and HIV, require fusionof the viral membrane with a host cell's membrane. For RSV, theconserved fusion protein (RSV-F glycoprotein) fuses the viral andcellular membranes by coupling irreversible protein refolding withjuxtaposition of the membranes. In current models based on paramyxovirusstudies, the RSV-F protein initially folds into a metastable“pre-fusion” conformation. During cell entry, the pre-fusionconformation undergoes refolding and conformational changes to itsstable “post-fusion” conformation. See, also, Swanson et al., PNAS USA108(23):9619-9624 (2011) regarding pre-fusion and post-fusion RSV-Fstructures.

In certain embodiments, the first and second antigens are from RSV. Forexample, the first and second antigens can independently be derived fromthe RSV surface glycoproteins Fusion (F), Glycoprotein (G), SmallHydrophobic protein (SH), the matrix proteins M and M2, the nucleocapsidproteins N, P and L, and the nonstructural proteins NS1 and NS2. Incertain preferred embodiments, the first and second antigens are each anRSV-F antigen.

The F glycoprotein of RSV is a type I single-pass integral membraneprotein having four general domains: N-terminal ER-translocating signalsequence (SS), ectodomain (ED), transmembrane domain (TM), and acytoplasmic tail (CT). CT contains a single palmitoylated cysteineresidue. The sequence of F protein is highly conserved among RSVisolates, but is constantly evolving (Kim et al. (2007) J Med Virol 79:820-828). Unlike most paramyxoviruses, the F protein in RSV can mediateentry and syncytium formation independent of the other viral proteins(HN is usually necessary in addition to F in other paramyxoviruses).

The RSV-F glycoprotein is translated from mRNA into an approximately 574amino acid protein designated F₀. Post-translational processing of F₀includes removal of an N-terminal signal peptide by a signal peptidasein the endoplasmic reticulum. F₀ is also cleaved at two sites(approximately 109/110 and approximately 136/137) by cellular proteases(in particular furin) in the trans-Golgi. This cleavage results in theremoval of a short intervening sequence and generates two subunitsdesignated F₁ (˜50 kDa; C-terminal; approximately residues 137-574) andF₂ (˜20 kDa; N-terminal; approximately residues 1-109) that remainassociated with each other. F₁ contains a hydrophobic fusion peptide atits N-terminus and also two amphipathic heptad-repeat regions (HRA andHRB). HRA is near the fusion peptide and HRB is near the transmembranedomain. Three F₁-F₂ heterodimers are assembled as homotrimers of F₁-F₂in the virion.

RSV-F antigens suitable for inclusion in the immunogenic compositionsdescribed herein, either in RNA encoded form or as polypeptides, includeRSV-F glycoprotein and RSV-F glycoprotein variants. Suitable RSV-Fglycoprotein variants include, for example, full length F protein andtruncated variants such as soluble ecto-domains, each optionallycontaining one or more mutations, such as furin-cleavage mutations,trypsin-cleavage mutations, fusion peptide mutations (e.g., deletions inwhole or in part), mutations that stabilize the HRB trimer, andmutations that destabilize the HRA trimer.

Full length and truncated RSV-F glycoprotiens, including those with oneor more such mutations in a variety of combinations are well known inthe art and are disclosed for example in WO2011/008974, the disclosureof which is incorporated herein by reference in its entirety.

The skilled addressee is directed to the following sections ofWO2011/008974 which disclose exemplary RSV-F antigens that can be used,in RNA form or as polypeptides, in the immunogenic compositions: (i)page 15, line 20 through page 16, line 27, which describes RSV-F, itsamino acid sequence and domain structure; (ii) page 16, line 28 throughpage 18, line 11, which describes soluble ectodomains of RSV-F; (iii)page 18, line 14 through page 20, line 15, which describesfurin-cleavage mutations, trypsin-cleavage mutations, fusion peptidemutations; (iv) page 20, line 16 through page 21, line 8, and page 26,line 29 through page 30, line 14, which describe optionaloligomerization sequences; (v) page 20, lines 9-24, which describeintroduced protease cleavage sites; (vi) and page 30, line 18 throughpage 32, line 18, which describe mutations that stabilize the HRBtrimer, destabilize the HRA trimer and other mutations that can beincluded.

In particular embodiments, the sequence of amino acid residue 100-150 ofthe RSV-F glycoprotein, such as SEQ ID NO:1 or SEQ ID NO:2 disclosed inWO2011/008974, or the soluble ecto domains thereof, is:

(Furmet) (SEQ ID NO: 25)TPATNNRARKELPRFMNYTLNNAKKTNVTLSKKRKKKFLGFLLGVGSAI AS (Furdel)(SEQ ID NO: 26) TPATNNRARQELPRFMNYTLNNAKKTNVTLSKK---RFLGELLGVGSAI AS(OFurex) (SEQ ID NO: 27)TPATNNQAQNELPQFMNYTLNNANNTNVTLSQNQNQNFLGFLLGVGSAI AS (NFurex)(SEQ ID NO: 28) TPATNNQAQNELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAI AS(Dp21Furex) (SEQ ID NO: 29)TPATNNQAQN---------------------QNQNQNFLGFLLGVGSAI AS (Dp23Fures)(SEQ ID NO: 30) TPATNNQAQN-----------------------QNQNFLGFLLGVGSAI AS(Dp23furdel) (SEQ ID NO: 31)TPATNNRARQ-----------------------QQQRFLGFLLGVGSAI AS (Nterm Furin)(SEQ ID NO: 32) TPATNNRARRELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAI AS(Cterm Furin) (SEQ ID NO: 33)TPATNNQAQNELPQFMNYTLNNAQQTNVTLSKKRKRRFLGFLLGVGSAI AS(Fusion peptide deletion 1) (SEQ ID NO: 34)TPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRR---------SAI AS,(Fusion peptide deletion 2) (SEQ ID NO: 35)TPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRR------GVGSAI AS,(Fusion peptide deletion 3) (SEQ ID NO: 36)TPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRR--------ASAI AS, or (Factor Xa)(SEQ ID NO: 37) TPATNNIEGRELPRFMNYTLNNAKKTNVILSKKIEGRFLGELLGVGSAI AS.In the foregoing sequences, the symbol “-” indicates that the amino acidat that position is deleted.

In particular embodiments, the sequence of the RSV-F antigen comprises:

RSV-E Fusion Deletion 1 Truncated HIS (SEQ ID NO: 38)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPREMNYTLNNAKKINVTLSKKRKRRSAIASGVAVSKVLELEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITRERSVNAGVITPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHISPLCTINIKEGSNICLTRTDRGWYCDNAGSVSFEPQAETCKVQSNRVECDTMNSLILPSEVNLCNVDIENPKYDCKIMTSKIDVSSSVITSLGAIVSCYGKIKCTASNKNRGIIKIFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHHHHHRSV-F Fusion Deletion 2 Truncated HIS (SEQ ID NO: 39)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKINVTLSKKRKRRGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLISKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNALLEITRERSVNAGVTIPVSTYMLINSELLSLINDMPIINDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCITNTKEGSNICLIRIDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHHHHHRSV-F Fusion Deletion 3 Truncated HIS (SEQ ID NO: 40)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRASAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLISKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNALLEITRERSVNAGVTIPVSTYMLINSELLSLINDMPIINDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCITNTKEGSNICLIRIDRGWYCDNAGSVSEFPQAETDKVQSNRVECDTMNSLTLPSEVNLDNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSDYGKTKDTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHHHHH

The sequences presented above contain a signal peptide and a HIS tag.The RSV-F protein used in the invention can contain any of the aminoacid sequences shown above, with or without the signal peptide and/orHIS tag.

Examples of additional RSV F antigens that can be used in the inventioninclude SEQ ID NOS: 6, 8 and 10 as disclosed in WO2009/079796 at pages58-60, which are incorporated herein by reference.

The RSV-F polypeptide suitable for inclusion in the immunogeniccompositions described herein may be in any desired form andconformation, including any desired mixture of forms and conformations.For example, the RSV-F polypeptide can be a monomer, or can be a trimercomprising three monomer polypeptides. Trimers can be monodispersed orcan be in the form of a rosette, for example, due to interactionsbetween the fusion peptides of individual timers. The immunogeniccompositions may comprise RSV-F polypeptides that are monomers, trimers,a combination of monomers and trimers (e.g., in dynamic equilibrium),rosettes of trimers, and any combination of the foregoing. The forms andconformations of RSV-F, including monomers, trimers, a combination ofmonomers and trimers (e.g., in dynamic equilibrium), rosettes oftrimers, cleaved and uncleaved forms, and pre-fusion and post-fusionforms are well known in the art, and are disclosed for example inWO2011/008974, the disclosure of which is incorporated herein byreference in its entirety. The skilled addressee is directedWO2011/008974, in particular at page 24, line 10 through page 26 line 27and Examples 2-7, which RSV-F proteins in a variety of forms andconformations and methods for making them.

2. CMV

In some aspects, the pathogen is CMV, and the first and second antigensare independently derived from CMV. In certain embodiments, the firstand second antigens are derived from a capsid protein, an envelopeglycoprotein (such as gB, gH, gL, gM, gN), or a tegument protein. Incertain embodiments, the first and second antigens are derived from oneor more of the following proteins: pp65, IE1, gB, gD, gH, gL, gM, gN,gO, UL128, UL129, gUL130, UL150, UL131, UL33, UL78, US27, US28, RL5A,RL6, RL10, RL11, RL12, RL13, UL1, UL2, UL4, UL5, UL6, UL7, UL8, UL9,UL10, UL11, UL14, UL15A, UL16, UL17, UL18, UL22A, UL38, UL40, UL41A,UL42, UL116, UL119, UL120, UL121, UL124, UL132, UL147A, UL148, UL142,UL144, UL141, UL140, UL135, UL136, UL138, UL139, UL133, UL135, UL148A,UL148B, UL148C, UL148D, US2, US3, US6, US7, US8, US9, US10, US11, US12,US13, US14, US15, US16, US17, US18, US19, US20, US21, US29, US30, orUS34A.

The CMV antigen may also be a fusion polypeptide of one or more CMVproteins, such as pp65/IE1 (Reap et al., Vaccine (2007) 25:7441-7449),gH/gL (Chowdary et al., Nature Structural & Molecular Biology, 17,882-888 (2010)).

Suitable CMV antigens include gB, gH, gL, gO, and can be from any CMVstrain. For example, CMV proteins can be from Merlin, AD169, VR1814,Towne, Toledo, TR, PH, TB40, or Fix strains of CMV. Exemplary sequencesof CMV proteins that may be used for the invention are shown in Table 1.

TABLE 1 Full length gH polynucleotide (CMV gH FL) SEQ ID NO: 7 Fulllength gH polypeptide (CMV gH FL) SEQ ID NO: 8 Full length gLpolynucleotide (CMV gL FL) SEQ ID NO: 11 Full length gL polypeptide (CMVgL FL) SEQ ID NO: 12 Full length gO polynucleotide (CMV gO FL) SEQ IDNO: 17 Full length gO polypeptide (CMV gO FL) SEQ ID NO: 18 gH solpolynucleotide (CMV gH sol) SEQ ID NO: 9 gH sol polypeptide (CMV gH sol)SEQ ID NO: 10 Full length UL128 polynucleotide (CMV UL128 FL) SEQ ID NO:19 Full length UL128 polypeptide (CMV UL128 FL) SEQ ID NO: 20 Fulllength UL130 polynucleotide (CMV UL130 FL) SEQ ID NO: 21 Full lengthUL130 polypeptide (CMV UL130 FL) SEQ ID NO: 22 Full length UL131polynucleotide (CMV UL131 FL) SEQ ID NO: 23 Full length UL131polypeptide (CMV UL131 FL) SEQ ID NO: 24 Full length gB polynucleotide(CMV gB FL) SEQ ID NO: 1 Full length gB polypeptide (CMV gB FL) SEQ IDNO: 2 gB sol 750 polynucleotide (CMV gB 750) SEQ ID NO: 3 gB sol 750polypeptide (CMV gB 750) SEQ ID NO: 4 gB sol 692 polynucleotide (CMV gB692) SEQ ID NO: 5 gB sol 692 polypeptide (CMV gB 692) SEQ ID NO: 6 Fulllength gM polynucleotide (CMV gM FL) SEQ ID NO: 13 Full length gMpolypeptide (CMV gM FL) SEQ ID NO: 14 Full length gN polynucleotide (CMVgN FL) SEQ ID NO: 15 Full length gN polypeptide (CMV gN FL) SEQ ID NO:16

gB Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a gB antigen. A gB antigen can be full length gB protein or canomit one or more regions of the protein. Alternatively, fragments of agB protein can be used. gB amino acids are numbered according to thefull-length gB amino acid sequence (CMV gB FL) shown in SEQ ID NO: 2,which is 907 amino acids long. Suitable regions of a gB protein, whichcan be excluded from the full-length protein or included as fragmentsinclude: the signal sequence (amino acids 1-24), a gB-DLDdisintegrin-like domain (amino acids 57-146), a furin cleavage site(amino acids 459-460), a heptad repeat region (679-693), a membranespanning domain (amino acids 751-771), and a cytoplasmic domain fromamino acids 771-906. In some embodiments, a gB antigen includes aminoacids 67-86 (Neutralizing Epitope AD2) and/or amino acids 532-635(Immunodominant Epitope AD1). Specific examples of gB antigens include“gB sol 692,” which includes the first 692 amino acids of gB, and “gBsol 750,” which includes the first 750 amino acids of gB. The signalsequence, amino acids 1-24, can be present or absent from gB sol 692 andgB sol 750 as desired.

In some embodiments, the gB antigen is a gB fragment of 10 amino acidsor longer. For example, the number of amino acids in the fragment cancomprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, or 875amino acids.

The invention may also use a gB antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 2 (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 2).

gH Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a gH antigen. A gH antigen can be a full-length gH protein (CMVgH FL, SEQ ID NO:8, for example, which is a 743 amino acid protein). gHhas a membrane spanning domain and a cytoplasmic domain starting atposition 716 to position 743. Removing amino acids from 717 to 743provides a soluble gH (e.g., CMV gH sol, SEQ ID NO: 10).

In some embodiments, the gH antigen is a gH fragment of 10 amino acidsor longer. For example, the number of amino acids in the fragment cancomprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, 700, or 725 amino acids.

The invention may also use a gH antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 8 or 10 (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO: 8 or 10).

gL Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a gL antigen. A gL antigen can be a full-length gL protein (CMVgL FL, SEQ ID NO:12, for example, which is a 278 amino acid protein).Alternatively, a gL fragment can be used. For example, the number ofamino acids in the fragment can comprise 10, 15, 20, 30, 40, 50, 60, 70,80, 90, 100, 125, 150, 175, 200, 225, or 250 amino acids.

The invention may also use a gL antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 12 (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 12).

gO Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a gO antigen. A gO antigen can be a full-length gO protein (CMVgO FL, SEQ ID NO:18, for example, which is a 472 amino acid protein).Alternatively, the gO antigen can be a gO fragment of 10 amino acids orlonger. For example, the number of amino acids in the fragment cancomprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, or 450 amino acids.

The invention may also use a gO antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 18 (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 18).

gM Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a gM antigen. A gM antigen can be a full-length gM protein (CMVgM FL, SEQ ID NO:14, for example, which is a 371 amino acid protein).Alternatively, the gM antigen can be a gM fragment of 10 amino acids orlonger. For example, the number of amino acids in the fragment cancomprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, or 350 amino acids.

The invention may also use a gM antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 14 (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 14).

gN Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a gN antigen. A gN antigen can be a full-length gN protein (CMVgN FL, SEQ ID NO:16, for example, which is a 135 amino acid protein).Alternatively, the gN antigen can be a gN fragment of 10 amino acids orlonger. For example, the number of amino acids in the fragment cancomprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125 aminoacids.

The invention may also use a gN antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 16 (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 16).

UL128 Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a UL128 antigen. A UL128 antigen can be a full-length UL128protein (CMV UL128 FL, SEQ ID NO:20, for example, which is a 171 aminoacid protein). Alternatively, the UL128 antigen can be a UL128 fragmentof 10 amino acids or longer. For example, the number of amino acids inthe fragment can comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,125, or 150 amino acids.

The invention may also use a UL128 antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 20 (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 20).

UL130 Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a UL130 antigen. A UL130 antigen can be a full-length UL130protein (CMV UL130 FL, SEQ ID NO:22, for example, which is a 214 aminoacid protein). Alternatively, the UL130 antigen can be a UL130 fragmentof 10 amino acids or longer. For example, the number of amino acids inthe fragment can comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, or 200 amino acids.

The invention may also use a UL130 antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 22 (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 22).

UL131 Antigens

In certain embodiments, the first antigen, the second antigen, or both,may be a UL131 antigen. A UL131 antigen can be a full-length UL131protein (CMV UL131, SEQ ID NO:24, for example, which is a 129 amino acidprotein). Alternatively, the UL131 antigen can be a UL131 fragment of 10amino acids or longer. For example, the number of amino acids in thefragment can comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125,150, 175, or 200 amino acids.

The invention may also use a UL131 antigen comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 24 (e.g., at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 24).

The CMV antigen may be a fusion polypeptide. For example, the antigenmay comprise a first domain and a second domain, wherein (i) the firstdomain comprises a first CMV antigen and (ii) the second domaincomprises a second CMV antigen. The first CMV antigen and the second CMVantigen are independently selected from a gB, gH, gL, gO, gM, gN, UL128,UL130, or UL131 antigen described above.

Two or more CMV antigens may also be co-delivered in the form of acomplex, or in a form that can form a complex in vivo (e.g., gH/gLcomplex, gM/gN complex, gH/gL/UL128/UL130/UL131 pentameric complex). Forexample, the immunogenic composition may comprise an RNA molecule thatencode two or more separate proteins, e.g, gH and gL. The immunogeniccomposition may also comprise two or more polypeptide antigens, e.g., gHand gL.

B. the RNA Molecule

The immunogenic composition described herein comprises an RNA componentand a polypeptide component. Preferably, the RNA is a self-replicatingRNA.

The composition can contain more than one RNA molecule encoding anantigen, e.g., two, three, five, ten or more RNA molecules.Alternatively or in addition, one RNA molecule may also encode more thanone antigen, e.g., a bicistronic, or tricistronic RNA molecule thatencodes different or identical antigens.

The sequence of the RNA molecule may be codon optimized or deoptimizedfor expression in a desired host, such as a human cell.

The sequence of the RNA molecule may be modified if desired, for exampleto increase the efficacy of expression or replication of the RNA, or toprovide additional stability or resistance to degradation. For example,the RNA sequence can be modified with respect to its codon usage, forexample, to increase translation efficacy and half-life of the RNA. Apoly A tail (e.g., of about 30 adenosine residues or more) may beattached to the 3′ end of the RNA to increase its half-life. The 5′ endof the RNA may be capped with a modified ribonucleotide with thestructure m7G (5′) ppp (5′) N (cap 0 structure) or a derivative thereof,which can be incorporated during RNA synthesis or can be enzymaticallyengineered after RNA transcription (e.g., by using Vaccinia VirusCapping Enzyme (VCE) consisting of mRNA triphosphatase,guanylyl-transferase and guanine-7-methyltransferase, which catalyzesthe construction of N7-monomethylated cap 0 structures). Cap 0 structureplays an important role in maintaining the stability and translationalefficacy of the RNA molecule. The 5′ cap of the RNA molecule may befurther modified by a 2′-O-Methyltransferase which results in thegeneration of a cap 1 structure (m7Gppp [m2′-O] N), which may furtherincreases translation efficacy.

If desired, the RNA molecule can comprise one or more modifiednucleotides in addition to any 5′ cap structure. There are more than 96naturally occurring nucleoside modifications found on mammalian RNA.See, e.g., Limbach et al., Nucleic Acids Research, 22(12):2183-2196(1994). The preparation of nucleotides and modified nucleotides andnucleosides are well-known in the art, e.g. from U.S. Pat. Nos.4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524,5,132,418, 5,153,319, 5,262,530, 5,700,642 all of which are incorporatedby reference in their entirety herein, and many modified nucleosides andmodified nucleotides are commercially available.

Modified nucleobases which can be incorporated into modified nucleosidesand nucleotides and be present in the RNA molecules include: m5C(5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U(2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A(2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A(2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A(2-methylthio-N6isopentenyladenosine); io6A(N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine);f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine);m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine);m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um (2-thio-2-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm (2′-O-methylinosine); m4C(N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G(N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm (5-formyl-2′-O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine)irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine),hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof,dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyluracil, 5-methyluracil, 5-(C₂-C₆)-alkenyluracil,5-(C₂-C₆)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil,5-fluorouracil, 5-bromouracil, 5-hydroxycytosine,5-(C₁-C₆)-alkylcytosine, 5-methylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 7-deazaguanine, 8-azaguanine,7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine,7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine,8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine,2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine,7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen(abasic residue), m5C, m5U, m6A, s2U, W, or 2′-O-methyl-U. Many of thesemodified nucleobases and their corresponding ribonucleosides areavailable from commercial suppliers. See, e.g., WO 2011/005799 which isincorporated herein by reference.

If desired, the RNA molecule can contain phosphoramidate,phosphorothioate, and/or methylphosphonate linkages.

In some embodiments, the RNA molecule does not include modifiednucleotides, e.g., does not include modified nucleobases, and all of thenucleotides in the RNA molecule are conventional standardribonucleotides A, U, G and C, with the exception of an optional 5′ capthat may include, for example, 7-methylguanosine. In other embodiments,the RNA may include a 5′ cap comprising a 7′-methylguanosine, and thefirst 1, 2 or 3 5′ ribonucleotides may be methylated at the 2′ positionof the ribose.

Self-Replicating RNA

In some aspects, the cationic oil in water emulsion contains aself-replicating RNA molecule. In certain embodiments, theself-replicating RNA molecule is derived from or based on an RNA virusor a retrovirus. In certain embodiments, the self-replicating RNAmolecule is derived from or based on an alphavirus.

Self-replicating RNA molecules are well known in the art and can beproduced by using replication elements derived from, e.g., alphaviruses,and substituting the structural viral proteins with a nucleotidesequence encoding a protein of interest. Cells transfected withself-replicating RNA briefly produce of antigen before undergoingapoptotic death. This death is a likely result of requisitedouble-stranded (ds) RNA intermediates, which also have been shown tosuper-activate Dendritic Cells. Thus, the enhanced immunogenicity ofself-replicating RNA may be a result of the production ofpro-inflammatory dsRNA, which mimics an RNA-virus infection of hostcells.

Advantageously, the cell's machinery is used by self-replicating RNAmolecules to generate an exponential increase of encoded gene products,such as proteins or antigens, which can accumulate in the cells or besecreted from the cells. Overexpression of proteins or antigens byself-replicating RNA molecules takes advantage of the immunostimulatoryadjuvant effects, including stimulation of toll-like receptors (TLR) 3,7 and 8 and non TLR pathways (e.g, RIG-1, MD-5) by the products of RNAreplication and amplification, and translation which induces apoptosisof the transfected cell.

The self-replicating RNA generally contains at least one or more genesselected from the group consisting of viral replicases, viral proteases,viral helicases and other nonstructural viral proteins, and alsocomprise 5′- and 3′-end cis-active replication sequences, and ifdesired, a heterologous sequences that encode a desired amino acidsequences (e.g., an antigen of interest). A subgenomic promoter thatdirects expression of the heterologous sequence can be included in theself-replicating RNA. If desired, the heterologous sequence (e.g., anantigen of interest) may be fused in frame to other coding regions inthe self-replicating RNA and/or may be under the control of an internalribosome entry site (IRES).

In certain embodiments, the self-replicating RNA molecule is notencapsulated in a virus-like particle. Self-replicating RNA molecules ofthe invention can be designed so that the self-replicating RNA moleculecannot induce production of infectious viral particles. This can beachieved, for example, by omitting one or more viral genes encodingstructural proteins that are necessary for the production of viralparticles in the self-replicating RNA. For example, when theself-replicating RNA molecule is based on an alpha virus, such asSinebis virus (SIN), Semliki forest virus and Venezuelan equineencephalitis virus (VEE), one or more genes encoding viral structuralproteins, such as capsid and/or envelope glycoproteins, can be omitted.

If desired, self-replicating RNA molecules of the invention can also bedesigned to induce production of infectious viral particles that areattenuated or virulent, or to produce viral particles that are capableof a single round of subsequent infection.

When delivered to a vertebrate cell, a self-replicating RNA molecule canlead to the production of multiple daughter RNAs by transcription fromitself (or from an antisense copy of itself). The self-replicating RNAcan be directly translated after delivery to a cell, and thistranslation provides a RNA-dependent RNA polymerase which then producestranscripts from the delivered RNA. Thus the delivered RNA leads to theproduction of multiple daughter RNAs. These transcripts are antisenserelative to the delivered RNA and may be translated themselves toprovide in situ expression of a gene product, or may be transcribed toprovide further transcripts with the same sense as the delivered RNAwhich are translated to provide in situ expression of the gene product.

One suitable system for achieving self-replication is to use analphavirus-based RNA replicon. Alphaviruses comprise a set ofgenetically, structurally, and serologically related arthropod-borneviruses of the Togaviridae family. Twenty-six known viruses and virussubtypes have been classified within the alphavirus genus, including,Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelanequine encephalitis virus. As such, the self-replicating RNA of theinvention may incorporate a RNA replicase derived from semliki forestvirus (SFV), sindbis virus (SIN), Venezuelan equine encephalitis virus(VEE), Ross-River virus (RRV), or other viruses belonging to thealphavirus family.

An alphavirus-based “replicon” expression vectors can be used in theinvention. Replicon vectors may be utilized in several formats,including DNA, RNA, and recombinant replicon particles. Such repliconvectors have been derived from alphaviruses that include, for example,Sindbis virus (Xiong et al. (1989) Science 243:1188-1191; Dubensky etal., (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol.72:950-958; Polo et al. (1999) PNAS 96:4598-4603), Semliki Forest virus(Liljestrom (1991) Bio/Technology 9:1356-1361; Berglund et al. (1998)Nat. Biotech. 16:562-565), and Venezuelan equine encephalitis virus(Pushko et al. (1997) Virology 239:389-401). Alphaviruses-derivedreplicons are generally quite similar in overall characteristics (e.g.,structure, replication), individual alphaviruses may exhibit someparticular property (e.g., receptor binding, interferon sensitivity, anddisease profile) that is unique. Therefore, chimeric alphavirusreplicons made from divergent virus families may also be useful.

Alphavirus-based replicons are (+)-stranded replicons that can betranslated after delivery to a cell to give of a replicase (orreplicase-transcriptase). The replicase is translated as a polyproteinwhich auto-cleaves to provide a replication complex which createsgenomic (−)-strand copies of the +-strand delivered RNA. These(−)-strand transcripts can themselves be transcribed to give furthercopies of the (+)-stranded parent RNA and also to give a subgenomictranscript which encodes the desired gene product. Translation of thesubgenomic transcript thus leads to in situ expression of the desiredgene product by the infected cell. Suitable alphavirus replicons can usea replicase from a sindbis virus, a semliki forest virus, an easternequine encephalitis virus, a venezuelan equine encephalitis virus, etc.

A preferred self-replicating RNA molecule thus encodes (i) aRNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) a polypeptide antigen. Thepolymerase can be an alphavirus replicase e.g. comprising alphavirusprotein nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non-structural replicase, it is preferred that analphavirus based self-replicating RNA molecule of the invention does notencode alphavirus structural proteins. Thus the self-replicating RNA canlead to the production of genomic RNA copies of itself in a cell, butnot to the production of RNA-containing alphavirus virions. Theinability to produce these virions means that, unlike a wild-typealphavirus, the self-replicating RNA molecule cannot perpetuate itselfin infectious form. The alphavirus structural proteins which arenecessary for perpetuation in wild-type viruses are absent fromself-replicating RNAs of the invention and their place is taken bygene(s) encoding the desired gene product, such that the subgenomictranscript encodes the desired gene product rather than the structuralalphavirus virion proteins.

Thus a self-replicating RNA molecule useful with the invention may havetwo open reading frames. The first (5′) open reading frame encodes areplicase; the second (3′) open reading frame encodes a polypeptideantigen. In some embodiments the RNA may have additional (downstream)open reading frames e.g. that encode another desired gene products. Aself-replicating RNA molecule can have a 5′ sequence which is compatiblewith the encoded replicase.

In other aspects, the self-replicating RNA molecule is derived from orbased on a virus other than an alphavirus, preferably, apositive-stranded RNA virus, and more preferably a picornavirus,flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, orcoronavirus. Suitable wild-type alphavirus sequences are well-known andare available from sequence depositories, such as the American TypeCulture Collection, Rockville, Md. Representative examples of suitablealphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCCVR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCCVR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCCVR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCCVR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus(ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580,ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCCVR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest(ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248),Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374),Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCCVR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis(ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCCVR-926), and Y-62-33 (ATCC VR-375).

The self-replicating RNA molecules of the invention are larger thanother types of RNA (e.g. mRNA). Typically, the self-replicating RNAmolecules of the invention contain at least about 4 kb. For example, theself-replicating RNA can contain at least about 5 kb, at least about 6kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, atleast about 10 kb, at least about 11 kb, at least about 12 kb or morethan 12 kb. In certain examples, the self-replicating RNA is about 4 kbto about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb,about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb toabout 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb,about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb toabout 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb,about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb toabout 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb,about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb toabout 11 kb.

The self-replicating RNA molecules of the invention may comprise one ormore modified nucleotides (e.g., pseudouridine, N6-methyladenosine,5-methylcytidine, 5-methyluridine).

The self-replicating RNA molecule may encode a single polypeptideantigen or, optionally, two or more of polypeptide antigens linkedtogether in a way that each of the sequences retains its identity (e.g.,linked in series) when expressed as an amino acid sequence. Thepolypeptides generated from the self-replicating RNA may then beproduced as a fusion polypeptide or engineered in such a manner toresult in separate polypeptide or peptide sequences.

The self-replicating RNA of the invention may encode one or morepolypeptide antigens that contain a range of epitopes. Preferablyepitopes capable of eliciting either a helper T-cell response or acytotoxic T-cell response or both.

The self-replicating RNA molecules described herein may be engineered toexpress multiple nucleotide sequences, from two or more open readingframes, thereby allowing co-expression of proteins, such as a two ormore antigens together with cytokines or other immunomodulators, whichcan enhance the generation of an immune response. Such aself-replicating RNA molecule might be particularly useful, for example,in the production of various gene products (e.g., proteins) at the sametime, for example, as a bivalent or multivalent vaccine.

The self-replicating RNA molecules of the invention can be preparedusing any suitable method. Several suitable methods are known in the artfor producing RNA molecules that contain modified nucleotides. Forexample, a self-replicating RNA molecule that contains modifiednucleotides can be prepared by transcribing (e.g., in vitrotranscription) a DNA that encodes the self-replicating RNA moleculeusing a suitable DNA-dependent RNA polymerase, such as T7 phage RNApolymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, and thelike, or mutants of these polymerases which allow efficientincorporation of modified nucleotides into RNA molecules. Thetranscription reaction will contain nucleotides and modifiednucleotides, and other components that support the activity of theselected polymerase, such as a suitable buffer, and suitable salts. Theincorporation of nucleotide analogs into a self-replicating RNA may beengineered, for example, to alter the stability of such RNA molecules,to increase resistance against RNases, to establish replication afterintroduction into appropriate host cells (“infectivity” of the RNA),and/or to induce or reduce innate and adaptive immune responses.

Suitable synthetic methods can be used alone, or in combination with oneor more other methods (e.g., recombinant DNA or RNA technology), toproduce a self-replicating RNA molecule of the invention. Suitablemethods for de novo synthesis are well-known in the art and can beadapted for particular applications. Exemplary methods include, forexample, chemical synthesis using suitable protecting groups such as CEM(Masuda et al., (2007) Nucleic Acids Symposium Series 51:3-4), theβ-cyanoethyl phosphoramidite method (Beaucage S L et al. (1981)Tetrahedron Lett 22:1859); nucleoside H-phosphonate method (Garegg P etal. (1986) Tetrahedron Lett 27:4051-4; Froehler B C et al. (1986) NuclAcid Res 14:5399-407; Garegg P et al. (1986) Tetrahedron Lett 27:4055-8;Gaffney B L et al. (1988) Tetrahedron Lett 29:2619-22). Thesechemistries can be performed or adapted for use with automated nucleicacid synthesizers that are commercially available. Additional suitablesynthetic methods are disclosed in Uhlmann et al. (1990) Chem Rev90:544-84, and Goodchild J (1990) Bioconjugate Chem 1: 165. Nucleic acidsynthesis can also be performed using suitable recombinant methods thatare well-known and conventional in the art, including cloning,processing, and/or expression of polynucleotides and gene productsencoded by such polynucleotides. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic polynucleotides areexamples of known techniques that can be used to design and engineerpolynucleotide sequences. Site-directed mutagenesis can be used to alternucleic acids and the encoded proteins, for example, to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, introduce mutations and the like.Suitable methods for transcription, translation and expression ofnucleic acid sequences are known and conventional in the art. (Seegenerally, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel,et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988;Glover, DNA Cloning, Vol. I, IRL Press, Wash., D.C., Ch. 3, 1986;Bitter, et al., in Methods in Enzymology 153:516-544 (1987); TheMolecular Biology of the Yeast Saccharomyces, Eds. Strathern et al.,Cold Spring Harbor Press, Vols. I and II, 1982; and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.)

The presence and/or quantity of one or more modified nucleotides in aself-replicating RNA molecule can be determined using any suitablemethod. For example, a self-replicating RNA can be digested tomonophosphates (e.g., using nuclease P1) and dephosphorylated (e.g.,using a suitable phosphatase such as CIAP), and the resultingnucleosides analyzed by reversed phase HPLC (e.g., usings a YMC PackODS-AQ column (5 micron, 4.6×250 mm) and elute using a gradient, 30% B(0-5 min) to 100% B (5-13 min) and at 100% B (13-40) min, flow Rate (0.7ml/min), UV detection (wavelength: 260 nm), column temperature (30° C.).Buffer A (20 mM acetic acid-ammonium acetate pH 3.5), buffer B (20 mMacetic acid-ammonium acetate pH 3.5/methanol [90/10])).

Optionally, the self-replicating RNA molecules of the invention mayinclude one or more modified nucleotides so that the self-replicatingRNA molecule will have less immunomodulatory activity upon introductionor entry into a host cell (e.g., a human cell) in comparison to thecorresponding self-replicating RNA molecule that does not containmodified nucleotides.

If desired, the self-replicating RNA molecules can be screened oranalyzed to confirm their therapeutic and prophylactic properties usingvarious in vitro or in vivo testing methods that are known to those ofskill in the art. For example, vaccines comprising self-replicating RNAmolecule can be tested for their effect on induction of proliferation oreffector function of the particular lymphocyte type of interest, e.g., Bcells, T cells, T cell lines, and T cell clones. For example, spleencells from immunized mice can be isolated and the capacity of cytotoxicT lymphocytes to lyse autologous target cells that contain a selfreplicating RNA molecule that encodes a polypeptide antigen. Inaddition, T helper cell differentiation can be analyzed by measuringproliferation or production of TH1 (IL-2 and IFN-γ) and/or TH2 (IL-4 andIL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmiccytokine staining and flow cytometry.

Self-replicating RNA molecules that encode a polypeptide antigen canalso be tested for ability to induce humoral immune responses, asevidenced, for example, by induction of B cell production of antibodiesspecific for an antigen of interest. These assays can be conductedusing, for example, peripheral B lymphocytes from immunized individuals.Such assay methods are known to those of skill in the art. Other assaysthat can be used to characterize the self-replicating RNA molecules ofthe invention can involve detecting expression of the encoded antigen bythe target cells. For example, FACS can be used to detect antigenexpression on the cell surface or intracellularly. Another advantage ofFACS selection is that one can sort for different levels of expression;sometimes-lower expression may be desired. Other suitable method foridentifying cells which express a particular antigen involve panningusing monoclonal antibodies on a plate or capture using magnetic beadscoated with monoclonal antibodies.

The self-replicating RNA of the invention may be delivered by a varietyof methods, such as naked RNA delivery or in combination with lipids,polymers or other compounds that facilitate entry into the cells. TheRNA molecules of the present invention can be introduced into targetcells or subjects using any suitable technique, e.g., by directinjection, microinjection, electroporation, lipofection, biolystics, andthe like.

C. The Polypeptide Molecule

The immunogenic composition described herein comprises a polypeptidecomponent and an RNA component. The polypeptide component encompassesmulti-chain polypeptide structures, such as a polypeptide complex (e.g.,a complex formed by two or more proteins), or a large polypeptidestructure, such as VLP.

Suitable antigens that can be used as the polypeptide component (the“second polypeptide antigen”) of the immunogenic composition includeproteins and peptides from any pathogen, such as a bacterial pathogen, aviral pathogen, a fungal pathogen, a protozoan pathogen, or amulti-cellular parasitic pathogen. Exemplary antigens include any one ofthe antigens described above, such as an antigen derived from RSV, HIV,or CMV. The composition can contain more than one polypeptide antigen.Alternatively or in addition, the polypeptide may also be a fusionpolypeptide comprising two or more epitopes from two different proteinsof the same pathogen, or two or more epitopes from two differentpathogens.

The polypeptide antigen may include additional sequences, such as asequence to facilitate expression, production, purification or detection(e.g., a poly-His sequence).

The polypeptide antigen will usually be isolated or purified. Thus, theywill not be associated with molecules with which they are normally, ifapplicable, found in nature.

Polypeptides will usually be prepared by expression in a recombinanthost system. Generally, they are produced by expression of recombinantconstructs that encode the ecto-domains in suitable recombinant hostcells, although any suitable methods can be used. Suitable recombinanthost cells include, for example, insect cells (e.g., Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni), mammalian cells (e.g., human,non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g.,hamster), avian cells (e.g., chicken, duck, and geese), bacteria (e.g.,E. coli, Bacillus subtilis, and Streptococcus spp.), yeast cells (e.g.,Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenualpolymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichiaguillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowialipolytica), Tetrahymena cells (e.g., Tetrahymena thermophila) orcombinations thereof. Many suitable insect cells and mammalian cells arewell-known in the art. Suitable insect cells include, for example, Sf9cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (aclonal isolate derived from the parental Trichoplusia ni BTI-TN-5B1-4cell line (Invitrogen)). Suitable mammalian cells include, for example,Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3cells, 293-T cells, Vero cells, HeLa cells, PERC.6 cells (ECACC depositnumber 96022940), Hep G2 cells, MRC-5 (ATCC CCI-171), WI-38 (ATCCCCL-75), fetal rhesus lung cells (ATCC CL-160), Madin-Darby bovinekidney (“MDBK”) cells, Madin-Darby canine kidney (“MDCK”) cells (e.g.,MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSM ACC 2219), baby hamsterkidney (BHK) cells, such as BHK21-F, HKCC cells, and the like. Suitableavian cells include, for example, chicken embryonic stem cells (e.g.,EBx® cells), chicken embryonic fibroblasts, chicken embryonic germcells, duck cells (e.g., AGE1.CR and AGE1.CR.pIX cell lines (ProBioGen)which are described, for example, in Vaccine 27:4975-4982 (2009) andWO2005/042728), EB66 cells, and the like.

Suitable insect cell expression systems, such as baculovirus systems,are known to those of skill in the art and described in, e.g., Summersand Smith, Texas Agricultural Experiment Station Bulletin No. 1555(1987). Materials and methods for baculovirus/insert cell expressionsystems are commercially available in kit form from, inter alia,Invitrogen, San Diego Calif. Avian cell expression systems are alsoknown to those of skill in the art and described in, e.g., U.S. Pat.Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; EuropeanPatent No. EP 0787180B; European Patent Application No. EP03291813.8; WO03/043415; and WO 03/076601. Similarly, bacterial and mammalian cellexpression systems are also known in the art and described in, e.g.,Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths,London.

Recombinant constructs encoding a polypeptide can be prepared insuitable vectors using conventional methods. A number of suitablevectors for expression of recombinant proteins in insect or mammaliancells are well-known and conventional in the art. Suitable vectors cancontain a number of components, including, but not limited to one ormore of the following: an origin of replication; a selectable markergene; one or more expression control elements, such as a transcriptionalcontrol element (e.g., a promoter, an enhancer, a terminator), and/orone or more translation signals; and a signal sequence or leadersequence for targeting to the secretory pathway in a selected host cell(e.g., of mammalian origin or from a heterologous mammalian ornon-mammalian species). For example, for expression in insect cells asuitable baculovirus expression vector, such as pFastBac (Invitrogen),is used to produce recombinant baculovirus particles. The baculovirusparticles are amplified and used to infect insect cells to expressrecombinant protein. For expression in mammalian cells, a vector thatwill drive expression of the construct in the desired mammalian hostcell (e.g., Chinese hamster ovary cells) is used.

Polypeptides can be purified using any suitable methods. For example,methods for purifying polypeptides by immunoaffinity chromatography areknown in the art. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687(2004). Suitable methods for purifying desired proteins includingprecipitation and various types of chromatography, such as hydrophobicinteraction, ion exchange, affinity, chelating and size exclusion arewell-known in the art. Suitable purification schemes can be createdusing two or more of these or other suitable methods. If desired, thepolypeptides can include a “tag” that facilitates purification, such asan epitope tag or a HIS tag. Such tagged polypeptides can convenientlybe purified, for example from conditioned media, by chelatingchromatography or affinity chromatography.

D. Optional RNA Delivery Systems

In addition to the protein component and the RNA component, additionalcomponents, such as lipids, polymers or other compounds may beoptionally included in the immunogenic composition as described hereinto facilitate the entry of RNA into target cells.

Although RNA can be delivered as naked RNA (e.g. merely as an aqueoussolution of RNA), to enhance entry into cells and also subsequentintercellular effects, the RNA molecule is preferably administered incombination with a delivery system, such as a particulate or emulsiondelivery system. A large number of delivery systems are well known tothose of skill in the art.

For example, the RNA molecule may be introduced into cells by way ofreceptor-mediated endocytosis. See e.g., U.S. Pat. No. 6,090,619; Wu andWu, J. Biol. Chem., 263:14621 (1988); and Curiel et al., Proc. Natl.Acad. Sci. USA, 88:8850 (1991). For example, U.S. Pat. No. 6,083,741discloses introducing an exogenous nucleic acid into mammalian cells byassociating the nucleic acid to a polycation moiety (e.g., poly-L-lysinehaving 3-100 lysine residues), which is itself coupled to an integrinreceptor-binding moiety (e.g., a cyclic peptide having the sequenceArg-Gly-Asp).

The RNA molecule of the present invention can be delivered into cellsvia amphiphiles. See e.g., U.S. Pat. No. 6,071,890. Typically, a nucleicacid molecule may form a complex with the cationic amphiphile. Mammaliancells contacted with the complex can readily take it up.

Three particularly useful delivery systems are (i) liposomes (ii)non-toxic and biodegradable polymer microparticles (iii) cationicsubmicron oil-in-water emulsions.

1. Liposomes

Various amphiphilic lipids can form bilayers in an aqueous environmentto encapsulate a RNA-containing aqueous core as a liposome. These lipidscan have an anionic, cationic or zwitterionic hydrophilic head group.Formation of liposomes from anionic phospholipids dates back to the1960s, and cationic liposome-forming lipids have been studied since the1990s. Some phospholipids are anionic whereas other are zwitterionic.Suitable classes of phospholipid include, but are not limited to,phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines,and phosphatidylglycerols, and some useful phospholipids are listed inTable 2. Useful cationic lipids include, but are not limited to,dioleoyl trimethylammonium propane (DOTAP),1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterioniclipids include, but are not limited to, acyl zwitterionic lipids andether zwitterionic lipids. Examples of useful zwitterionic lipids areDPPC, DOPC and dodecylphosphocholine. The lipids can be saturated orunsaturated.

TABLE 2 Phospholipids DDPC1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DLOPC1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA1,2-Dilauroyl-sn-Glycero-3-Phosphate DLPC1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine DLPG1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DLPS1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine DMG1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine DMPA1,2-Dimyristoyl-sn-Glycero-3-Phosphate DMPC1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine DMPG1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DMPS1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine DOPA1,2-Dioleoyl-sn-Glycero-3-Phosphate DOPC1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine DOPG1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DOPS1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine DPPA1,2-Dipalmitoyl-sn-Glycero-3-Phosphate DPPC1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine DPPG1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DPPS1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine DPyPE1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA1,2-Distearoyl-sn-Glycero-3-Phosphate DSPC1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE1,2-Distearpyl-sn-Glycero-3-phosphatidylethanolamine DSPG1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DSPS1,2-Distearoyl-sn-Glycero-3-phosphatidylserine EPC Egg-PC HEPCHydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPCHydrogenated Soy PC LYSOPC MYRISTIC1-Myristoyl-sn-Glycero-3-phosphatidylcholine LYSOPC PALMITIC1-Palmitoyl-sn-Glycero-3-phosphatidylcholine LYSOPC STEARIC1-Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin MPPC1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholine MSPC1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine PMPC1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine POPC1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine POPE1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine POPG1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol) . . .] PSPC1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine SMPC1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine SOPC1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine SPPC1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine

Liposomes can be formed from a single lipid or from a mixture of lipids.A mixture may comprise (i) a mixture of anionic lipids (ii) a mixture ofcationic lipids (iii) a mixture of zwitterionic lipids (iv) a mixture ofanionic lipids and cationic lipids (v) a mixture of anionic lipids andzwitterionic lipids (vi) a mixture of zwitterionic lipids and cationiclipids or (vii) a mixture of anionic lipids, cationic lipids andzwitterionic lipids. Similarly, a mixture may comprise both saturatedand unsaturated lipids. For example, a mixture may comprise DSPC(zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/or DMPG(anionic, saturated). Where a mixture of lipids is used, not all of thecomponent lipids in the mixture need to be amphiphilic e.g. one or moreamphiphilic lipids can be mixed with cholesterol.

The hydrophilic portion of a lipid can be PEGylated (i.e. modified bycovalent attachment of a polyethylene glycol). This modification canincrease stability and prevent non-specific adsorption of the liposomes.For instance, lipids can be conjugated to PEG using techniques such asthose disclosed in Heyes et al. (2005) J Controlled Release 107:276-87.

A mixture of DSPC, DlinDMA, PEG-DMPG and cholesterol is used in theexamples. A separate aspect of the invention is a liposome comprisingDSPC, DlinDMA, PEG-DMG and cholesterol. This liposome preferablyencapsulates RNA, such as a self-replicating RNA e.g. encoding animmunogen.

Liposomes are usually divided into three groups: multilamellar vesicles(MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles(LUV). MLVs have multiple bilayers in each vesicle, forming severalseparate aqueous compartments. SUVs and LUVs have a single bilayerencapsulating an aqueous core; SUVs typically have a diameter ≤50 nm,and LUVs have a diameter >50 nm. Liposomes useful with of the inventionare ideally LUVs with a diameter in the range of 50-220 nm. For acomposition comprising a population of LUVs with different diameters:(i) at least 80% by number should have diameters in the range of 20-220nm, (ii) the average diameter (Zav, by intensity) of the population isideally in the range of 40-200 nm, and/or (iii) the diameters shouldhave a polydispersity index <0.2.

Techniques for preparing suitable liposomes are well known in the arte.g. see Liposomes: Methods and Protocols, Volume 1: PharmaceuticalNanocarriers: Methods and Protocols. (ed. Weissig). Humana Press, 2009.ISBN 160327359X; Liposome Technology, volumes I, II & III. (ed.Gregoriadis). Informa Healthcare, 2006; and Functional Polymer Colloidsand Microparticles volume 4 (Microspheres, microcapsules & liposomes).(eds. Arshady & Guyot). Citus Books, 2002. One useful method involvesmixing (i) an ethanolic solution of the lipids (ii) an aqueous solutionof the nucleic acid and (iii) buffer, followed by mixing, equilibration,dilution and purification (Heyes et al. (2005) J Controlled Release107:276-87).

RNA is preferably encapsulated within the liposomes, and so the liposomeforms a outer layer around an aqueous RNA-containing core. Thisencapsulation has been found to protect RNA from RNase digestion. Theliposomes can include some external RNA (e.g. on the surface of theliposomes), but at least half of the RNA (and ideally all of it) isencapsulated.

2. Polymeric Microparticles

Various polymers can form microparticles to encapsulate or adsorb RNA.The use of a substantially non-toxic polymer means that a recipient cansafely receive the particles, and the use of a biodegradable polymermeans that the particles can be metabolised after delivery to avoidlong-term persistence. Useful polymers are also sterilisable, to assistin preparing pharmaceutical grade formulations.

Suitable non-toxic and biodegradable polymers include, but are notlimited to, poly(α-hydroxy acids), polyhydroxy butyric acids,polylactones (including polycaprolactones), polydioxanones,polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates,tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones orpolyester-amides, and combinations thereof.

In some embodiments, the microparticles are formed from poly(α-hydroxyacids), such as a poly(lactides) (“PLA”), copolymers of lactide andglycolide such as a poly(D,L-lactide-co-glycolide) (“PLG”), andcopolymers of D,L-lactide and caprolactone. Useful PLG polymers includethose having a lactide/glycolide molar ratio ranging, for example, from20:80 to 80:20 e.g. 25:75, 40:60, 45:55, 55:45, 60:40, 75:25. Useful PLGpolymers include those having a molecular weight between, for example,5,000-200,000 Da e.g. between 10,000-100,000, 20,000-70,000,40,000-50,000 Da.

The microparticles ideally have a diameter in the range of 0.02 μm to 8μm. For a composition comprising a population of microparticles withdifferent diameters at least 80% by number should have diameters in therange of 0.03-7 μm.

Techniques for preparing suitable microparticles are well known in theart e.g. see Functional Polymer Colloids and Microparticles volume 4(Microspheres, microcapsules & liposomes). (eds. Arshady & Guyot). CitusBooks, 2002; Polymers in Drug Delivery. (eds. Uchegbu & Schatzlein). CRCPress, 2006. (in particular chapter 7) and Microparticulate Systems forthe Delivery of Proteins and Vaccines. (eds. Cohen & Bernstein). CRCPress, 1996. To facilitate adsorption of RNA, a microparticle mayinclude a cationic surfactant and/or lipid e.g. as disclosed in O'Haganet al. (2001) J Virology 75:9037-9043; and Singh et al. (2003)Pharmaceutical Research 20: 247-251. An alternative way of makingpolymeric microparticles is by molding and curing e.g. as disclosed inWO2009/132206.

Microparticles of the invention can have a zeta potential of between40-100 mV.

RNA can be adsorbed to the microparticles, and adsorption is facilitatedby including cationic materials (e.g. cationic lipids) in themicroparticle.

3. Oil-In-Water Cationic Emulsions

Oil-in-water emulsions are known for adjuvanting influenza vaccines e.g.the MF59™ adjuvant in the FLUAD™ product, and the AS03 adjuvant in thePREPANDRIX™ product. RNA delivery according to the present invention canutilise an oil-in-water emulsion, provided that the emulsion includesone or more cationic molecules. For instance, a cationic lipid can beincluded in the emulsion to provide a positive droplet surface to whichnegatively-charged RNA can attach.

The emulsion comprises one or more oils. Suitable oil(s) include thosefrom, for example, an animal (such as fish) or a vegetable source. Theoil is ideally biodegradable (metabolisable) and biocompatible. Sourcesfor vegetable oils include nuts, seeds and grains. Peanut oil, soybeanoil, coconut oil, and olive oil, the most commonly available, exemplifythe nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean.Seed oils include safflower oil, cottonseed oil, sunflower seed oil,sesame seed oil and the like. In the grain group, corn oil is the mostreadily available, but the oil of other cereal grains such as wheat,oats, rye, rice, teff, triticale and the like may also be used. 6-10carbon fatty acid esters of glycerol and 1,2-propanediol, while notoccurring naturally in seed oils, may be prepared by hydrolysis,separation and esterification of the appropriate materials starting fromthe nut and seed oils. Fats and oils from mammalian milk aremetabolizable and so may be used. The procedures for separation,purification, saponification and other means necessary for obtainingpure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Squalene can also be obtained from yeast or other suitable microbes. Insome embodiments, Squalene is preferably obtained from non-animalsources, such as from olives, olive oil or yeast. Squalane, thesaturated analog to squalene, can also be used. Fish oils, includingsqualene and squalane, are readily available from commercial sources ormay be obtained by methods known in the art.

Other useful oils are the tocopherols, particularly in combination withsqualene. Where the oil phase of an emulsion includes a tocopherol, anyof the α, β, γ, δ, ε or ξ tocopherols can be used, but α-tocopherols arepreferred. D-α-tocopherol and DL-α-tocopherol can both be used. Apreferred α-tocopherol is DL-α-tocopherol. An oil combination comprisingsqualene and a tocopherol (e.g. DL-α-tocopherol) can be used.

Preferred emulsions comprise squalene, a shark liver oil which is abranched, unsaturated terpenoid (C₃₀H₅₀;[(CH₃)₂C[═CHCH₂CH₂C(CH₃)]₂═CHCH₂—]₂;2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN7683-64-9).

The oil in the emulsion may comprise a combination of oils e.g. squaleneand at least one further oil.

The aqueous component of the emulsion can be plain water (e.g. w.f.i.)or can include further components e.g. solutes. For instance, it mayinclude salts to form a buffer e.g. citrate or phosphate salts, such assodium salts. Typical buffers include: a phosphate buffer, a Trisbuffer, a borate buffer, a succinate buffer, a histidine buffer, or acitrate buffer. A buffered aqueous phase is preferred, and buffers willtypically be included in the 5-20 mM range.

The emulsion also includes a cationic lipid. Preferably this lipid is asurfactant so that it can facilitate formation and stabilisation of theemulsion. Useful cationic lipids generally contains a nitrogen atom thatis positively charged under physiological conditions e.g. as a tertiaryor quaternary amine. This nitrogen can be in the hydrophilic head groupof an amphiphilic surfactant. Useful cationic lipids include, but arenot limited to: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),3′-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecyl-ammonium (DDA e.g. the bromide),1,2-Dimyristoyl-3-Trimethyl-AmmoniumPropane (DMTAP),dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA). Other usefulcationic lipids are: benzalkonium chloride (BAK), benzethonium chloride,cetramide (which contains tetradecyltrimethylammonium bromide andpossibly small amounts of dedecyltrimethylammonium bromide andhexadecyltrimethyl ammonium bromide), cetylpyridinium chloride (CPC),cetyl trimethylammonium chloride (CTAC), primary amines, secondaryamines, tertiary amines, including but not limited toN,N′,N′-polyoxyethylene (10)-N-tallow-1,3-diaminopropane, otherquaternary amine salts, including but not limited tododecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium bromide,mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammoniumchloride, benzyldimethylhexadecyl-ammonium chloride,benzyltrimethylammonium methoxide, cetyldimethylethylammonium bromide,dimethyldioctadecyl ammonium bromide (DDAB), methylbenzethoniumchloride, decamethonium chloride, methyl mixed trialkyl ammoniumchloride, methyl trioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemetha-naminiumchloride (DEBDA), dialkyldimetylammonium salts,[1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio) propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl,distearoyl, dioleoyl),1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl3-succinyl-sn-glycerol choline ester, cholesteryl (4′-trimethylammonio)butanoate), N-alkyl pyridinium salts (e.g. cetylpyridinium bromide andcetylpyridinium chloride), N-alkylpiperidinium salts, dicationicbolaform electrolytes (C₁₂Me₆; C₁₂Bu₆),dialkylglycetylphosphorylcholine, lysolecithin, L-αdioleoylphosphatidylethanolamine, cholesterol hemisuccinate cholineester, lipopolyamines, including but not limited todioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine(LPLL, LPDL), poly (L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C₁₂GluPhC_(n)N⁺), ditetradecyl glutamate ester withpendant amino group (C₁₄GluC_(n)N⁺), cationic derivatives ofcholesterol, including but not limited to cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3β-oxysuccinamidoethylene dimethylamine, cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt, cholesteryl-3β-carboxyamidoethylenedimethylamine. Other useful cationic lipids aredescribed in US 2008/0085870 and US 2008/0057080, which are incorporatedherein by reference.

The cationic lipid is preferably biodegradable (metabolisable) andbiocompatible.

In addition to the oil and cationic lipid, an emulsion can include anon-ionic surfactant and/or a zwitterionic surfactant. Such surfactantsinclude, but are not limited to: the polyoxyethylene sorbitan esterssurfactants (commonly referred to as the Tweens), especially polysorbate20 and polysorbate 80; copolymers of ethylene oxide (EO), propyleneoxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™tradename, such as linear EO/PO block copolymers; octoxynols, which canvary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, withoctoxynol-9 (TRITON™ X-100 surfactant, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); polyoxyethylene fatty ethersderived from lauryl, cetyl, stearyl and oleyl alcohols (known as BRIJ®surfactants), such as triethyleneglycol monolauryl ether (BRIJ® 30surfactant); polyoxyethylene-9-lauryl ether; and sorbitan esters(commonly known as the Spans), such as sorbitan trioleate (SPAN® 85surfactant) and sorbitan monolaurate. Preferred surfactants forincluding in the emulsion are polysorbate 80 (TWEEN® 80 surfactant;polyoxyethylene sorbitan monooleate), SPAN® 85 surfactant (sorbitantrioleate), lecithin and TRITON™ X-100 surfactant.

Mixtures of these surfactants can be included in the emulsion e.g.TWEEN® 80/SPAN® 85 mixtures, or TWEEN® 80/TRITON™-X100 surfactantmixtures. A combination of a polyoxyethylene sorbitan ester such aspolyoxyethylene sorbitan monooleate (TWEEN® 80 surfactant) and anoctoxynol such as t-octylphenoxy-polyethoxyethanol (TRITON™ X-100surfactant) is also suitable. Another useful combination compriseslaureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.Useful mixtures can comprise a surfactant with a HLB value in the rangeof 10-20 (e.g. polysorbate 80, with a HLB of 15.0) and a surfactant witha HLB value in the range of 1-10 (e.g. sorbitan trioleate, with a HLB of1.8).

Preferred amounts of oil (% by volume) in the final emulsion are between2-20% e.g. 5-15%, 6-14%, 7-13%, 8-12%. A squalene content of about 4-6%or about 9-11% is particularly useful.

Preferred amounts of surfactants (% by weight) in the final emulsion arebetween 0.001% and 8%. For example: polyoxyethylene sorbitan esters(such as polysorbate 80) 0.2 to 4%, in particular between 0.4-0.6%,between 0.45-0.55%, about 0.5% or between 1.5-2%, between 1.8-2.2%,between 1.9-2.1%, about 2%, or 0.85-0.95%, or about 1%; sorbitan esters(such as sorbitan trioleate) 0.02 to 2%, in particular about 0.5% orabout 1%; octyl- or nonylphenoxy polyoxyethanols (such as TRITON™ X-100surfactant) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethyleneethers (such as laureth 9) 0.1 to 8%, preferably 0.1 to 10% and inparticular 0.1 to 1% or about 0.5%.

The absolute amounts of oil and surfactant, and their ratio, can bevaried within wide limits while still forming an emulsion. A skilledperson can easily vary the relative proportions of the components toobtain a desired emulsion, but a weight ratio of between 4:1 and 5:1 foroil and surfactant is typical (excess oil).

An important parameter for ensuring immunostimulatory activity of anemulsion, particularly in large animals, is the oil droplet size(diameter). The most effective emulsions have a droplet size in thesubmicron range. Suitably the droplet sizes will be in the range 50-750nm. Most usefully the average droplet size is less than 250 nm e.g. lessthan 200 nm, less than 150 nm. The average droplet size is usefully inthe range of 80-180 nm. Ideally, at least 80% (by number) of theemulsion's oil droplets are less than 250 nm in diameter, and preferablyat least 90%. Apparatuses for determining the average droplet size in anemulsion, and the size distribution, are commercially available. Thesethese typically use the techniques of dynamic light scattering and/orsingle-particle optical sensing e.g. the Accusizer™ and Nicomp™ seriesof instruments available from Particle Sizing Systems (Santa Barbara,USA), or the Zetasizer™ instruments from Malvern Instruments (UK), orthe Particle Size Distribution Analyzer instruments from Horiba (Kyoto,Japan).

Ideally, the distribution of droplet sizes (by number) has only onemaximum i.e. there is a single population of droplets distributed aroundan average (mode), rather than having two maxima. Preferred emulsionshave a polydispersity of <0.4 e.g. 0.3, 0.2, or less.

Suitable emulsions with submicron droplets and a narrow sizedistribution can be obtained by the use of microfluidisation. Thistechnique reduces average oil droplet size by propelling streams ofinput components through geometrically fixed channels at high pressureand high velocity. These streams contact channel walls, chamber wallsand each other. The results shear, impact and cavitation forces cause areduction in droplet size. Repeated steps of microfluidisation can beperformed until an emulsion with a desired droplet size average anddistribution are achieved.

As an alternative to microfluidisation, thermal methods can be used tocause phase inversion. These methods can also provide a submicronemulsion with a tight particle size distribution.

Preferred emulsions can be filter sterilised i.e. their droplets canpass through a 220 nm filter. As well as providing a sterilisation, thisprocedure also removes any large droplets in the emulsion.

In certain embodiments, the cationic lipid in the emulsion is DOTAP. Thecationic oil-in-water emulsion may comprise from about 0.5 mg/ml toabout 25 mg/ml DOTAP. For example, the cationic oil-in-water emulsionmay comprise DOTAP at from about 0.5 mg/ml to about 25 mg/ml, from about0.6 mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml,from about 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, from about 0.5mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8 mg/ml, fromabout 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml to about 1.6mg/ml, from about 0.6 mg/ml to about 1.6 mg/ml, from about 0.7 mg/ml toabout 1.6 mg/ml, from about 0.8 mg/ml to about 1.6 mg/ml, about 0.5mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 12mg/ml, about 18 mg/ml, about 20 mg/ml, about 21.8 mg/ml, about 24 mg/ml,etc. In an exemplary embodiment, the cationic oil-in-water emulsioncomprises from about 0.8 mg/ml to about 1.6 mg/ml DOTAP, such as 0.8mg/ml, 1.2 mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DC Cholesterol. Thecationic oil-in-water emulsion may comprise DC Cholesterol at from about0.1 mg/ml to about 5 mg/ml DC Cholesterol. For example, the cationicoil-in-water emulsion may comprise DC Cholesterol from about 0.1 mg/mlto about 5 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3mg/ml to about 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, fromabout 0.5 mg/ml to about 5 mg/ml, from about 0.62 mg/ml to about 5mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1.5 mg/ml toabout 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about 2.46mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, from about3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml, fromabout 4.5 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.92mg/ml, from about 0.1 mg/ml to about 4.5 mg/ml, from about 0.1 mg/ml toabout 4 mg/ml, from about 0.1 mg/ml to about 3.5 mg/ml, from about 0.1mg/ml to about 3 mg/ml, from about 0.1 mg/ml to about 2.46 mg/ml, fromabout 0.1 mg/ml to about 2 mg/ml, from about 0.1 mg/ml to about 1.5mg/ml, from about 0.1 mg/ml to about 1 mg/ml, from about 0.1 mg/ml toabout 0.62 mg/ml, about 0.15 mg/ml, about 0.3 mg/ml, about 0.6 mg/ml,about 0.62 mg/ml, about 0.9 mg/ml, about 1.2 mg/ml, about 2.46 mg/ml,about 4.92 mg/ml, etc. In an exemplary embodiment, the cationicoil-in-water emulsion comprises from about 0.62 mg/ml to about 4.92mg/ml DC Cholesterol, such as 2.46 mg/ml.

In certain embodiments, the cationic lipid is DDA. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 5 mg/mlDDA. For example, the cationic oil-in-water emulsion may comprise DDA atfrom about 0.1 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.5mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg/ml toabout 3.5 mg/ml, from about 0.1 mg/ml to about 3 mg/ml, from about 0.1mg/ml to about 2.5 mg/ml, from about 0.1 mg/ml to about 2 mg/ml, fromabout 0.1 mg/ml to about 1.5 mg/ml, from about 0.1 mg/ml to about 1.45mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml toabout 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, from about 0.5mg/ml to about 5 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, fromabout 0.73 mg/ml to about 5 mg/ml, from about 0.8 mg/ml to about 5mg/ml, from about 0.9 mg/ml to about 5 mg/ml, from about 1.0 mg/ml toabout 5 mg/ml, from about 1.2 mg/ml to about 5 mg/ml, from about 1.45mg/ml to about 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about2.5 mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, fromabout 3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml,from about 4.5 mg/ml to about 5 mg/ml, about 1.2 mg/ml, about 1.45mg/ml, etc. Alternatively, the cationic oil-in-water emulsion maycomprise DDA at about 20 mg/ml, about 21 mg/ml, about 21.5 mg/ml, about21.6 mg/ml, about 25 mg/ml. In an exemplary embodiment, the cationicoil-in-water emulsion comprises from about 0.73 mg/ml to about 1.45mg/ml DDA, such as 1.45 mg/ml.

The RNA molecules of the invention can also be delivered to cells exvivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by re-implantation of the cells intoa patient, usually after selection for cells which have been transfectedwith the RNA molecule. The appropriate amount of cells to deliver to apatient will vary with patient conditions, and desired effect, which canbe determined by a skilled artisan. See e.g., U.S. Pat. Nos. 6,054,288;6,048,524; and 6,048,729. Preferably, the cells used are autologous,i.e., cells obtained from the patient being treated.

E. Adjuvants

In certain embodiments, the immunogenic compositions provided hereininclude or optionally include one or more immunoregulatory agents suchas adjuvants. Exemplary adjuvants include, but are not limited to, a TH1adjuvant and/or a TH2 adjuvant, further discussed below. In certainembodiments, the adjuvants used in the immunogenic compositions provideherein include, but are not limited to:

-   -   1. Mineral-Containing Compositions;    -   2. Oil Emulsions;    -   3. Saponin Formulations;    -   4. Virosomes and Virus-Like Particles;    -   5. Bacterial or Microbial Derivatives;    -   6. Bioadhesives and Mucoadhesives;    -   7. Liposomes;    -   8. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations;    -   9. Polyphosphazene (PCPP);    -   10. Muramyl Peptides;    -   11. Imidazoquinolone Compounds;    -   12. Thiosemicarbazone Compounds;    -   13. Tryptanthrin Compounds;    -   14. Human Immunomodulators;    -   15. Lipopeptides;    -   16. Benzonaphthyridines;    -   17. Microparticles    -   18. Immunostimulatory polynucleotide (such as RNA or DNA; e.g.,        CpG-containing oligonucleotides)

1. Mineral Containing Compositions

Mineral containing compositions suitable for use as adjuvants includemineral salts, such as aluminum salts and calcium salts. The immunogeniccomposition may include mineral salts such as hydroxides (e.g.,oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates),sulfates, etc. (see, e.g., VACCINE DESIGN: THE SUBUNIT AND ADJUVANTAPPROACH (Powell, M. F. and Newman, M J. eds.) (New York: Plenum Press)1995, Chapters 8 and 9), or mixtures of different mineral compounds(e.g. a mixture of a phosphate and a hydroxide adjuvant, optionally withan excess of the phosphate), with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption to thesalt(s) being preferred. The mineral containing compositions may also beformulated as a particle of metal salt (WO 00/23105).

Aluminum salts may be included in vaccines of the invention such thatthe dose of Al³⁺ is between 0.2 and 1.0 mg per dose.

In certain embodiments, the aluminum based adjuvant is alum (aluminumpotassium sulfate (AlK(SO₄)₂), or an alum derivative, such as thatformed in-situ by mixing an antigen in phosphate buffer with alum,followed by titration and precipitation with a base such as ammoniumhydroxide or sodium hydroxide.

Another aluminum-based adjuvant suitable for use in vaccine formulationsis aluminum hydroxide adjuvant (Al(OH)₃) or crystalline aluminumoxyhydroxide (AlOOH), which is an excellent adsorbant, having a surfacearea of approximately 500 m²/g. Alternatively, the aluminum basedadjuvant can be aluminum phosphate adjuvant (AlPO₄) or aluminumhydroxyphosphate, which contains phosphate groups in place of some orall of the hydroxyl groups of aluminum hydroxide adjuvant. Preferredaluminum phosphate adjuvants provided herein are amorphous and solublein acidic, basic and neutral media.

In certain embodiments, the adjuvant comprises both aluminum phosphateand aluminum hydroxide. In a more particular embodiment, the adjuvanthas a greater amount of aluminum phosphate than aluminum hydroxide, suchas a ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or greater than9:1, by weight aluminum phosphate to aluminum hydroxide. In anotherembodiment, aluminum salts in the vaccine are present at 0.4 to 1.0 mgper vaccine dose, or 0.4 to 0.8 mg per vaccine dose, or 0.5 to 0.7 mgper vaccine dose, or about 0.6 mg per vaccine dose.

Generally, the preferred aluminum-based adjuvant(s), or ratio ofmultiple aluminum-based adjuvants, such as aluminum phosphate toaluminum hydroxide is selected by optimization of electrostaticattraction between molecules such that the antigen carries an oppositecharge as the adjuvant at the desired pH. For example, aluminumphosphate adjuvant (iep=4) adsorbs lysozyme, but not albumin at pH 7.4.Should albumin be the target, aluminum hydroxide adjuvant would beselected (iep=4). Alternatively, pretreatment of aluminum hydroxide withphosphate lowers its isoelectric point, making it a preferred adjuvantfor more basic antigens.

2. Oil-Emulsions

Oil-emulsion compositions and formulations suitable for use as adjuvants(with or without other specific immunostimulating agents such as muramylpeptides or bacterial cell wall components) include squalene-wateremulsions, such as MF59 (5% Squalene, 0.5% TWEEN® 80, and 0.5% SPAN® 85,formulated into submicron particles using a microfluidizer). See WO90/14837. See also, Podda (2001) VACCINE 19: 2673-2680; Frey et al.(2003) Vaccine 21:4234-4237. MF59 is used as the adjuvant in the FLUAD™influenza virus trivalent subunit vaccine.

Particularly preferred oil-emulsion adjuvants for use in thecompositions are submicron oil-in-water emulsions. Preferred submicronoil-in-water emulsions for use herein are squalene/water emulsionsoptionally containing varying amounts of MTP-PE, such as a submicronoil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v TWEEN®80™ surfactant (polyoxyethylenesorbitan monooleate), and/or 0.25-1.0%SPAN® 85™ surfactant (sorbitan trioleate), and, optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(l′-2′-dipalmitoyl-SM-glycero-3-huydroxyphosphophoryloxy)-ethylamine(MTP-PE), for example, the submicron oil-in-water emulsion known as“MF59” (WO 90/14837; U.S. Pat. Nos. 6,299,884; 6,451,325; and Ott etal., “MF59—Design and Evaluation of a Safe and Potent Adjuvant for HumanVaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell,M. F. and Newman, M J. eds.) (New York: Plenum Press) 1995, pp.277-296). MF59 contains 4-5% w/v Squalene (e.g. 4.3%), 0.25-0.5% w/vTWEEN® 80™ surfactant, and 0.5% w/v SPAN® 85™ surfactant and optionallycontains various amounts of MTP-PE, formulated into submicron particlesusing a microfluidizer such as Model 11OY microfluidizer (Microfluidics,Newton, Mass.). For example, MTP-PE may be present in an amount of about0-500 μg/dose, more preferably 0-250 μg/dose and most preferably, 0-100μg/dose. As used herein, the term “MF59-0” refers to the above submicronoil-in-water emulsion lacking MTP-PE, while the term MF59-MTP denotes aformulation that contains MTP-PE. For instance, “MF59-100” contains 100μg MTP-PE per dose, and so on. MF69, another submicron oil-in-wateremulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v TWEEN®80™ surfactant, and 0.75% w/v SPAN® 85™ surfactant and optionallyMTP-PE. Yet another submicron oil-in-water emulsion is MF75, also knownas SAF, containing 10% squalene, 0.4% TWEEN® 80™ surfactant, 5%pluronic-blocked polymer L121, and thr-MDP, also microfluidized into asubmicron emulsion. MF75-MTP denotes an MF75 formulation that includesMTP, such as from 100-400 μg MTP-PE per dose.

Submicron oil-in-water emulsions, methods of making the same andimmunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in WO 90/14837; U.S. Pat. Nos.6,299,884; and 6,451,325.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)may also be used as adjuvants in the invention.

3. Other Immunological Adjuvants

Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponins isolated from thebark of the Quillaia saponaria Molina tree have been widely studied asadjuvants. Saponins can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. Saponin adjuvant formulations include STIMULON® adjuvant(Antigenics, Inc., Lexington, Mass.).

Saponin compositions have been purified using High Performance ThinLayer Chromatography (HP-TLC) and Reversed Phase High Performance LiquidChromatography (RP-HPLC). Specific purified fractions using thesetechniques have been identified, including QS7, QS 17, QS 18, QS21,QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method ofproduction of QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponinformulations may also comprise a sterol, such as cholesterol (see WO96/33739).

Saponin formulations may include sterols, cholesterols and lipidformulations. Combinations of saponins and cholesterols can be used toform unique particles called Immunostimulating Complexes (ISCOMs).ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of Quil A,QHA and QHC. ISCOMs are further described in EP 0 109 942, WO 96/11711and WO 96/33739. Optionally, the ISCOMS may be devoid of (an) additionaldetergent(s). See WO 00/07621.

A review of the development of saponin based adjuvants can be found inBarr et al. (1998) ADV. DRUG DEL. REV. 32:247-271. See also Sjolander etal. (1998) ADV. DRUG DEL. REV. 32:321-338.

Virosomes and Virus Like Particles (VLPs) generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein pi). VLPs are discussed furtherin WO 03/024480; WO 03/024481; Niikura et al. (2002) VIROLOGY293:273-280; Lenz et al. (2001) J. IMMUNOL. 166(9):5346-5355′ Pinto etal. (2003) J. INFECT. DIS. 188:327-338; and Gerber et al. (2001) J.VIROL. 75(10):4752-4760. Virosomes are discussed further in, forexample, Gluck et al. (2002) VACCINE 20:B10-B16. Immunopotentiatingreconstituted influenza virosomes (IRIV) are used as the subunit antigendelivery system in the intranasal trivalent INFLEXAL™ product (Mischlerand Metcalfe (2002) VACCINE 20 Suppl 5:B17-B23) and the INFLUVAC PLUS™product.

Bacterial or microbial derivatives suitable for use as adjuvantsinclude, but are not limited to:

(1) Non-toxic derivatives of enterobacterial lipopolysaccharide (LPS):Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylatedMPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipidA with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such“small particles” of 3dMPL are small enough to be sterile filteredthrough a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPSderivatives include monophosphoryl lipid A mimics, such as aminoalkylglucosaminide phosphate derivatives, e.g., RC-529. See Johnson et al.(1999) Bioorg. Med. Chem. Lett. 9:2273-2278.

(2) Lipid A Derivatives: Lipid A derivatives include derivatives oflipid A from Escherichia coli such as OM-174. OM-174 is described forexample in Meraldi et al. (2003) Vaccine 21:2485-2491; and Pajak et al.(2003) Vaccine 21:836-842. Another exemplary adjuvant is the syntheticphospholipid dimer, E6020 (Eisai Co. Ltd., Tokyo, Japan), which mimicsthe physicochemical and biological properties of many of the naturallipid A's derived from Gram-negative bacteria.

(3) Immunostimulatory oligonucleotides: Immunostimulatoryoligonucleotides or polymeric molecules suitable for use as adjuvants inthe invention include nucleotide sequences containing a CpG motif (asequence containing an unmethylated cytosine followed by guanosine andlinked by a phosphate bond). Bacterial double stranded RNA oroligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory. The CpG's can include nucleotidemodifications/analogs such as phosphorothioate modifications and can bedouble-stranded or single-stranded. Optionally, the guanosine may bereplaced with an analog such as 2′-deoxy-7-deazaguanosine. SeeKandimalla et al. (2003) Nucl. Acids Res. 31(9): 2393-2400; WO 02/26757;and WO 99/62923 for examples of possible analog substitutions. Theadjuvant effect of CpG oligonucleotides is further discussed in Krieg(2003) Nat. Med. 9(7):831-835; McCluskie et al. (2002) FEMS Immunol.Med. Microbiol. 32: 179-185; WO 98/40100; U.S. Pat. Nos. 6,207,646;6,239,116; and 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT. See Kandimalla et al. (2003) Biochem. Soc. Trans. 31 (part3):654-658. The CpG sequence may be specific for inducing a ThI immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inBlackwell et al. (2003) J. Immunol. 170(8):4061-4068; Krieg (2002)TRENDS Immunol. 23(2): 64-65; and WO 01/95935. Preferably, the CpG is aCpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, Kandimalla et al. (2003) BBRC 306:948-953; Kandimalla etal. (2003) Biochem. Soc. Trans. 31 (part 3):664-658′ Bhagat et al.(2003) BBRC 300:853-861; and WO03/035836.

Immunostimulatory oligonucleotides and polymeric molecules also includealternative polymer backbone structures such as, but not limited to,polyvinyl backbones (Pitha et al. (1970) Biochem. Biophys. Acta204(1):39-48; Pitha et al. (1970) Biopolymers 9(8):965-977), andmorpholino backbones (U.S. Pat. Nos. 5,142,047; 5,185,444). A variety ofother charged and uncharged polynucleotide analogs are known in the art.Numerous backbone modifications are known in the art, including, but notlimited to, uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, and carbamates) and charged linkages(e.g., phosphorothioates and phosphorodithioates).

Adjuvant IC31, Intercell AG, Vienna, Austria, is a synthetic formulationthat contains an antimicrobial peptide, KLK, and an immunostimulatoryoligonucleotide, ODNIa. The two component solution may be simply mixedwith antigens (e.g., particles in accordance with the invention with anassociated antigen), with no conjugation required.

ADP-ribosylating toxins and detoxified derivatives thereof: BacterialADP-ribosylating toxins and detoxified derivatives thereof may be usedas adjuvants in the invention. Preferably, the protein is derived fromE. coli (i.e., E. coli heat labile enterotoxin “LT”), cholera (“CT”), orpertussis (“PT”). The use of detoxified ADP-ribosylating toxins asmucosal adjuvants is described in WO 95/17211 and as parenteraladjuvants in WO 98/42375. Preferably, the adjuvant is a detoxified LTmutant such as LT-K63, LT-R72, and LTR192G. The use of ADP-ribosylatingtoxins and detoxified derivatives thereof, particularly LT-K63 andLT-R72, as adjuvants can be found in the following references: Beignonet al. (2002) Infect. Immun. 70(6):3012-3019; Pizza et al. (2001)Vaccine 19:2534-2541; Pizza et al. (2000) J. Med. Microbiol.290(4-5):455-461; Scharton-Kersten et al. (2000) Infect. Immun.68(9):5306-5313′ Ryan et al. (1999) Infect. Immun. 67(12):6270-6280;Partidos et al. (1999) Immunol. Lett. 67(3):209-216; Peppoloni et al.(2003) Vaccines 2(2):285-293; and Pine et al. (2002) J. Control Release85(1-3):263-270. Numerical reference for amino acid substitutions ispreferably based on the alignments of the A and B subunits ofADP-ribosylating toxins set forth in Domenighini et al. (1995) MoI.Microbiol. 15(6): 1165-1167.

Bioadhesives and mucoadhesives may also be used as adjuvants. Suitablebioadhesives include esterified hyaluronic acid microspheres (Singh etal. (2001) J. Cont. Release 70:267-276) or mucoadhesives such ascross-linked derivatives of polyacrylic acid, polyvinyl alcohol,polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose.Chitosan and derivatives thereof may also be used as adjuvants in theinvention (see WO 99/27960).

Examples of liposome formulations suitable for use as adjuvants aredescribed in U.S. Pat. Nos. 6,090,406; 5,916,588; and EP PatentPublication No. EP 0 626 169.

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters (see, e.g., WO 99/52549). Suchformulations further include polyoxyethylene sorbitan ester surfactantsin combination with an octoxynol (WO 01/21207) as well aspolyoxyethylene alkyl ethers or ester surfactants in combination with atleast one additional non-ionic surfactant such as an octoxynol (WO01/21152). Preferred polyoxyethylene ethers are selected from thefollowing group: polyoxyethylene-9-lauryl ether (laureth 9),polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether.

PCPP formulations suitable for use as adjuvants are described, forexample, in Andrianov et al. (1998) Biomaterials 19(1-3): 109-115; andPayne et al. (1998) Adv. Drug Del. Rev. 31(3): 185-196.

Examples of muramyl peptides suitable for use as adjuvants includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), andN-acetylmuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2-(l′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

Examples of imidazoquinoline compounds suitable for use as adjuvantsinclude Imiquimod and its analogues, which are described further inStanley (2002) Clin. Exp. Dermatol. 27(7):571-577; Jones (2003) Curr.Opin. Investig. Drugs 4(2):214-218; and U.S. Pat. Nos. 4,689,338;5,389,640; 5,268,376; 4,929,624; 5,266,575; 5,352,784; 5,494,916;5,482,936; 5,346,905; 5,395,937; 5,238,944; and 5,525,612.

Examples of thiosemicarbazone compounds suitable for use as adjuvants,as well as methods of formulating, manufacturing, and screening for suchcompounds, include those described in WO 04/60308. Thethiosemicarbazones are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

Examples of tryptanthrin compounds suitable for use as adjuvants, aswell as methods of formulating, manufacturing, and screening for suchcompounds, include those described in WO 04/64759. The tryptanthrincompounds are particularly effective in the stimulation of humanperipheral blood mononuclear cells for the production of cytokines, suchas TNF-α. examples of benzonaphthyridine compounds suitable for use asadjuvants include:

Examples of benzonaphthyridine compounds suitable for use as adjuvants,as well as methods of formulating and manufacturing, include thosedescribed in WO 2009/111337.

Lipopeptides suitable for use as adjuvants are described above. Otherexemplary lipopeptides include, e.g., LP 40, which is an agonist ofTLR2. See, e.g., Akdis, et al, EUR. J. IMMUNOLOGY, 33: 2717-26 (2003).Murein lipopeptides are lipopeptides derived from E. coli. See, Hantke,et al., Eur. J. Biochem., 34: 284-296 (1973). Murein lipopeptidescomprise a peptide linked to N-acetyl muramic acid, and are thus relatedto Muramyl peptides, which are described in Baschang, et al.,Tetrahedron, 45(20): 6331-6360 (1989).

The human immunomodulators suitable for use as adjuvants include, butare not limited to, cytokines, such as, by way of example only,interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12), interferons(such as, by way of example only, interferon-γ), macrophage colonystimulating factor, and tumor necrosis factor.

Microparticles suitable for use as adjuvants include, but are notlimited to, microparticles formed from materials that are biodegradableand non-toxic (e.g. a poly(.alpha.-hydroxy acid), a polyhydroxybutyricacid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide). In certain embodiments, such microparticlesare treated to have a negatively-charged surface (e.g. with SDS) or apositively-charged surface (e.g. with a cationic detergent, such asCTAB). The microparticles suitable for use as adjuvants have a particlediameter of about 100 nm to about 150 μm in diameter. In certainembodiments, the particle diameter is about 200 nm to about 30 μm, andin other embodiments the particle diameter is about 500 nm to 10 μm.

3. Kits

(A) Kits for Co-Administration of an RNA Molecule and a PolypeptideMolecule

The invention also provides kits, wherein an RNA molecule encoding afirst polypeptide antigen (the RNA component); and a second polypeptideantigen (the polypeptide component), are in separate containers. Forexample, the kit can contain a first container comprising a compositioncomprising an RNA molecule encoding a first polypeptide antigen, and asecond container comprising a composition comprising a secondpolypeptide antigen. The polypeptide or the RNA molecule can be inliquid form or can be in solid form (e.g., lyophilized).

The kits described may be used for co-delivery of the RNA component andthe polypeptide component of the immunogenic compositions describedherein (e.g., the RNA component and the polypeptide component are mixedprior to administration for simultaneous delivery, e.g., mixed withinabout 72 hours, about 48 hours, about 24 hours, about 12 hours, about 10hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour,about 45 minutes, about 30 minutes, about 15 minutes, about 10 minutes,or about 5 minutes prior to administration).

(B) Kits for Prime-Boost

In another aspect, the invention provides a kit comprising: (i) apriming composition comprising a self-replicating RNA molecule thatencodes a first polypeptide antigen that comprises a first epitope; and(ii) a boosting composition comprising a second polypeptide antigen thatcomprises a second epitope; wherein said first epitope and secondepitope are the same epitope. The kits are suitable for sequentialadministration of the RNA and the polypeptide, such as a “RNA prime,protein boost” immunization regimen to generate an immune response to apathogen.

Suitable antigens that can be used as the RNA-coded antigen (the firstpolypeptide antigen) for the priming composition, or the polypeptideantigen (the second polypeptide antigen) for the boosting compositioninclude proteins and peptides from any pathogen, such as a bacterialpathogen, a viral pathogen, a fungal pathogen, a protozoan pathogen, ora multi-cellular parasitic pathogen. Exemplary antigens include any oneof the antigens described above, such as an antigen derived from RSV,HIV, or CMV.

The RNA molecule of the priming composition can be delivered as nakedRNA (e.g. merely as an aqueous solution of RNA). Alternatively, toenhance entry into cells and also subsequent intercellular effects, thepriming composition may optionally comprise a delivery system (such as aparticulate or emulsion delivery system), so that the RNA molecule isadministered in combination with the delivery system. Exemplary deliverysystems are described above. The delivery system may be in the samecontainer as the RNA molecule (e.g., pre-formulated), or in a differentcontainer from the RNA (e.g., the RNA and the delivery system areseparately packaged, and may be combined, e.g., within about 72 hours,about 48 hours, about 24 hours, about 12 hours, about 10 hours, about 9hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about4 hours, about 3 hours, about 2 hours, about 1 hour, about 45 minutes,about 30 minutes, about 15 minutes, about 10 minutes, or about 5 minutesprior to administration).

The priming composition, the boosting composition, or both, mayoptionally include one or more immunoregulatory agents such asadjuvants, as described herein. The immunoregulatory agent may be in thesame container as the priming or boosting composition, or in a separatecontained that can be combined with the priming or boosting compositionprior to administration.

The priming composition comprising the RNA molecule or the boostingcomposition comprising the polypeptide can be in liquid form or can bein solid form (e.g., lyophilized).

(C) Other Components of the Kits

Suitable containers include, for example, bottles, vials, syringes, andtest tubes. Containers can be formed from a variety of materials,including glass or plastic. A container may have a sterile access port(for example, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle).

The kit can further comprise a third container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. It can also contain othermaterials useful to the end-user, including other pharmaceuticallyacceptable formulating solutions such as buffers, diluents, filters,needles, and syringes or other delivery device. The kit may furtherinclude a fourth container comprising an adjuvant (such as an aluminumcontaining adjuvant or MF59).

The kit can also comprise a package insert containing writteninstructions for methods of inducing immunity or for treatinginfections. The package insert can be an unapproved draft package insertor can be a package insert approved by the Food and Drug Administration(FDA) or other regulatory body.

The invention also provides a delivery device pre-filled with theimmunogenic compositions, the priming compositions, or the boostingcompositions described above.

4. Pharmaceutical Compositions

In one aspect, the invention relates to pharmaceutical compositionscomprising an RNA component and a polypeptide component. Thepharmaceutical composition comprises: (i) a self-replicating RNAmolecule that encodes a first polypeptide antigen comprising a firstepitope (the RNA component); and (ii) a second polypeptide antigencomprising a second epitope (the polypeptide component); wherein saidfirst epitope and second epitope are epitopes from the same pathogen;and (iii) a pharmaceutically acceptable carrier and/or apharmaceutically acceptable vehicle.

In another aspect, the invention relates to a kit comprising: (i) apriming composition comprising a self-replicating RNA molecule thatencodes a first polypeptide antigen that comprises a first epitope; and(ii) a boosting composition comprising a second polypeptide antigen thatcomprises a second epitope; wherein said first epitope and secondepitope are the same epitope; and wherein the priming composition, theboosting composition, or both, comprise(s) a pharmaceutically acceptablecarrier and/or a pharmaceutically acceptable vehicle.

The pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier and/or a suitable delivery system as described herein(such as liposomes, nanoemulsions, PLG micro- and nanoparticles,lipoplexes, chitosan micro- and nanoparticles and other polyplexes forRNA delivery). If desired other pharmaceutically acceptable componentscan be included, such as excipients and adjuvants. These pharmaceuticalcompositions can be used as vaccines.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. A variety of aqueous carriers can be used. Suitablepharmaceutically acceptable carriers for use in the pharmaceuticalcompositions include plain water (e.g. w.f.i.) or a buffer e.g. aphosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, ahistidine buffer, or a citrate buffer. Buffer salts will typically beincluded in the 5-20 mM range.

The pharmaceutical compositions are preferably sterile, and may besterilized by conventional sterilization techniques.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, and tonicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like.

Preferably, the pharmaceutical compositions of the invention may have apH between 5.0 and 9.5, e.g. between 6.0 and 8.0.

Pharmaceutical compositions of the invention may include sodium salts(e.g. sodium chloride) to give tonicity. A concentration of 10±2 mg/mlNaCl is typical e.g. about 9 mg/ml.

Pharmaceutical compositions of the invention may have an osmolarity ofbetween 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, orbetween 290-310 mOsm/kg.

Pharmaceutical compositions of the invention may include one or morepreservatives, such as thiomersal or 2-phenoxyethanol. Mercury-freecompositions are preferred, and preservative-free vaccines can beprepared.

Pharmaceutical compositions of the invention are preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose. Pharmaceutical compositionsof the invention are preferably gluten free.

The concentrations of the polypeptide molecule and/or the RNA moleculein the pharmaceutical compositions can vary, and will be selected basedon fluid volumes, viscosities, body weight and other considerations inaccordance with the particular mode of administration selected and theintended recipient's needs. However, the pharmaceutical compositions areformulated to provide an effective amount of RNA+polypeptide (eitheradministered simultaneously, or administered sequentially, such as RNAprime, protein boost), such as an amount (either in a single dose or aspart of a series) that is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, age, the taxonomic group of individual to betreated (e.g. non-human primate, primate, etc.), the capacity of theindividual's immune system to react to the antigen encoded protein orpeptide, the condition to be treated, and other relevant factors. It isexpected that the amount will fall in a relatively broad range that canbe determined through routine trials. The RNA content of compositionswill generally be expressed in terms of the amount of RNA per dose. Apreferred dose has ≤200 μg, ≤100 μg, ≤50 μg, or ≤10 μg RNA, andexpression can be seen at much lower levels e.g. ≤1 μg/dose, ≤100ng/dose, ≤10 ng/dose, ≤1 ng/dose, etc. The amount of polypeptide in eachdose will generally comprise from about 0.1 to about 100 μg ofpolypeptide, with from about 5 to about 50 μg being preferred and fromabout 5 to about 25 μg/dose being alternatively preferred.

The amount of adjuvant, if any, will be an amount that will induce animmunomodulating response without significant adverse side effect. Anoptional amount for a particular vaccine can be ascertained by standardstudies involving observation of a vaccine's antibody titers and theirvirus neutralization capabilities. The amount of adjuvant will be fromabout 1 to about 100 μg/dose, with from about 5 to about 50 μg/dosebeing preferred, and from about 20 to about 50 μg/dose beingalternatively preferred.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous orintraperitoneal injection, and preferably by intramuscular, intradermalor subcutaneous injection, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampoulesand vials. Injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets. Cells transduced by the RNAmolecules can also be administered intravenously or parenterally.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, tragacanth, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, carriersknown in the art.

It is recognized that polypeptide and RNA molecules, when administeredorally, must be protected from digestion. Protection of polypeptide andRNA molecules can typically be accomplished either by complexing the RNAmolecule or the polypeptide molecule with a composition to render theRNA/polypeptide resistant to acidic and enzymatic hydrolysis, or bypackaging the RNA molecule or the polypeptide molecule in anappropriately resistant carrier such as a liposome. Means of protectingnucleic acids (such as RNA molecules) and polypeptides from digestionare well known in the art.

The pharmaceutical compositions can be encapsulated, e.g., in liposomes,or in a formulation that provides for slow release of the activeingredient. For example, the RNA molecule may be formulated asliposomes, then administered as a priming composition. Alternatively,liposome-formulated RNA may be mixed with the polypeptide molecule toproduce the RNA+polypeptide immunogenic composition of the invention.Alternatively, the RNA molecule and the polypeptide molecule can beco-encapsulated in liposomes.

The compositions described herein (priming compositions, boostingcompositions, or immunogenic compositions comprising an RNA and apolypeptide), alone or in combination with other suitable components,can be made into aerosol formulations (e.g., they can be “nebulized”) tobe administered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Suitable suppository formulations may contain the RNA, the polypeptide,or the polypeptide and RNA combination as described herein, and asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. It is also possible touse gelatin rectal capsules filled with the polypeptide and RNAmolecules as described herein, and a suitable base, for example, liquidtriglycerides, polyethylene glycols, and paraffin hydrocarbons.

5. Methods of Generating or Enhancing Immune Responses

(A) Co-Administration of an RNA Molecule and a Polypeptide Molecule

In another aspect, the invention provides a method for inducing,generating or enhancing an immune response in a subject in need thereof,such as a vertebrate, preferably a mammal, comprising administering aneffective amount of an immunogenic composition comprising an RNAcomponent and a polypeptide component. The composition comprises: (i) aself-replicating RNA molecule that encodes a first polypeptide antigencomprising a first epitope (the RNA component); and (ii) a secondpolypeptide antigen comprising a second epitope (the polypeptidecomponent); wherein said first epitope and second epitope are epitopesfrom the same pathogen. The immune response is preferably protective andpreferably involves antibodies and/or cell-mediated immunity. The methodmay be used to induce a primary immune response and/or to boost animmune response.

In another aspect, the immunogenic compositions disclosed herein may beused in the manufacture of a medicament for inducing, generating, orenhancing an immune response in a subject in need thereof, such as avertebrate, preferably a mammal.

In another aspect, the invention provides a method for treating orpreventing an infectious disease in a subject (such as a vertebrate,preferably a mammal) in need thereof, comprising administering aneffective amount of an immunogenic composition comprising an RNAcomponent and a polypeptide component. The composition comprises: (i) aself-replicating RNA molecule that encodes a first polypeptide antigencomprising a first epitope (the RNA component); and (ii) a secondpolypeptide antigen comprising a second epitope (the polypeptidecomponent); wherein said first epitope and second epitope are epitopesfrom the same pathogen.

In another aspect, the compositions disclosed herein may be used in themanufacture of a medicament for treating or preventing an infectiousdisease in a subject in need thereof, such as a vertebrate, preferably amammal.

In another aspect, the invention provides a method for vaccinating asubject, such as a vertebrate, preferably a mammal, or immunizing asubject against a pathogen (e.g., a bacterial pathogen, a viralpathogen, a fungal pathogen, a protozoan pathogen, or a multi-cellularparasitic pathogen), comprising administering to a subject in needthereof an effective amount of an immunogenic composition comprising anRNA component and a polypeptide component. The composition comprises:(i) a self-replicating RNA molecule that encodes a first polypeptideantigen comprising a first epitope (the RNA component); and (ii) asecond polypeptide antigen comprising a second epitope (the polypeptidecomponent); wherein said first epitope and second epitope are epitopesfrom the same pathogen.

In another aspect, the compositions disclosed herein may be used in themanufacture of a medicament for vaccinating a subject in need thereof,such as a vertebrate, preferably a mammal.

When the RNA molecule and the polypeptide molecule are co-administered,it may still be desirable to package the polypeptide molecule and RNAmolecule separately. The two components may be combined, e.g., withinabout 72 hours, about 48 hours, about 24 hours, about 12 hours, about 10hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour,about 45 minutes, about 30 minutes, about 15 minutes, about 10 minutes,or about 5 minutes prior to administration. For example, the polypeptidemolecule and RNA molecule can be combined at a patient's bedside.

(B) Prime-Boost

One aspect of the invention relates to the “prime and boost”immunization regimes in which the immune response induced by a primingcomposition is boosted by a boosting composition. For example, followingpriming (at least once) with an antigen (e.g., a polypeptide antigen, anRNA-coded antigen, an attenuated pathogen, or a combination thereof), aboosting composition comprising substantially the same antigen in thesame form (e.g., protein prime, protein boost; RNA prime, RNA boost;etc.), substantially the same antigen in a different form (e.g., RNAprime, protein boost; in which the RNA and the protein are directed tothe same target antigen), or a different antigen in the same or adifferent form (e.g., RNA prime targeting antigen 1, protein boosttargeting antigen 2, wherein antigen 1 and antigen 2 are different butshare a common epitope), may be administered to boost the immuneresponse in the primed host.

In another aspect, the invention provides a method for inducing,generating or enhancing an immune response in a subject in need thereof,such as a vertebrate, preferably a mammal, comprising: (i) administeringto a subject in need thereof at least once a therapeutically effectiveamount of a priming composition comprising a self-replicating RNAmolecule that encodes a first polypeptide antigen that comprises a firstepitope; and (ii) subsequently administering the subject at least once atherapeutically effective amount of a boosting composition comprising asecond polypeptide antigen that comprises a second epitope; wherein saidfirst epitope and second epitope are the same epitope. The immuneresponse is preferably protective and preferably involves antibodiesand/or cell-mediated immunity.

In another aspect, the priming and boosting compositions disclosedherein may be used in the manufacture of a medicament for inducing,generating, or enhancing an immune response in a subject in needthereof, such as a vertebrate, preferably a mammal.

In another aspect, the invention provides a method for treating orpreventing an infectious disease in a subject (such as a vertebrate,preferably a mammal) in need thereof, comprising: (i) administering to asubject in need thereof at least once a therapeutically effective amountof a priming composition comprising a self-replicating RNA molecule thatencodes a first polypeptide antigen that comprises a first epitope; and(ii) subsequently administering the subject at least once atherapeutically effective amount of a boosting composition comprising asecond polypeptide antigen that comprises a second epitope; wherein saidfirst epitope and second epitope are the same epitope.

In another aspect, the priming and boosting compositions disclosedherein may be used in the manufacture of a medicament for treating orpreventing an infectious disease in a subject in need thereof, such as avertebrate, preferably a mammal.

In another aspect, the invention provides a method for vaccinating asubject, such as a vertebrate, preferably a mammal, or immunizing asubject against a pathogen (e.g., a bacterial pathogen, a viralpathogen, a fungal pathogen, a protozoan pathogen, or a multicellularparasitic pathogen), comprising: (i) administering to a subject in needthereof at least once a therapeutically effective amount of a primingcomposition comprising a self-replicating RNA molecule that encodes afirst polypeptide antigen that comprises a first epitope; and (ii)subsequently administering the subject at least once a therapeuticallyeffective amount of a boosting composition comprising a secondpolypeptide antigen that comprises a second epitope; wherein said firstepitope and second epitope are the same epitope.

In another aspect, the priming and boosting compositions disclosedherein may be used in the manufacture of a medicament for vaccinating asubject in need thereof, such as a vertebrate, preferably a mammal.

The priming composition and the boosting composition may besubstantially the same (e.g., RNA+protein prime, RNA+protein boost), ormay be different (e.g., RNA+protein prime, protein boost).

The antigens (either in polypeptide form or in RNA-coded form) to beincluded in the priming and boosting compositions need not be identical,but should share at least one common epitope (e.g., the primingcomposition comprising an RNA molecule that encodes a first polypeptideantigen that comprises a first epitope; the boosting compositioncomprising a second polypeptide antigen that comprises a second epitope;wherein said first epitope and second epitope are the same epitope).

One embodiment of the invention uses an “RNA prime, protein boost”immunization strategy. Following priming (at least once) with an RNAmolecule, a polypeptide molecule is subsequently administered to boostthe immune response in the primed host.

Another embodiment of the invention uses an “RNA+protein prime, proteinboost” strategy. Following priming (at least once) with an immunogeniccomposition comprising an RNA molecule and a polypeptide molecule, apolypeptide molecule is subsequently administered to boost the immuneresponse in the primed host.

The subject may be primed and/or boosted more than once. For example,the immunization strategy can be prime, prime, boost; or prime, boost,boost. In certain embodiment, the priming composition is administered asleast twice, at least 3 times, at least 4 times, or at least 5 times. Incertain embodiment, the boost composition is administered as leasttwice, at least 3 times, at least 4 times, or at least 5 times.

Administration of the boosting composition is generally weeks or monthsafter administration of the priming composition, such as about 1 week,about 2 weeks, about 3 weeks, about 4 weeks, about 8 weeks, about 12weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks,about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48weeks, about 52 weeks, about 1 month, about 2 months, about 3 months,about 4 months, about 5 months, about 6 months, about 7 months, about 8months, about 9 months, about 10 months, about 11 months, about 12months, about 18 months, about 2 years, about 3 years, about 4 years,about 5 years, about 6 years, about 7 years, about 8 years, about 9years, or about 10 years after the priming composition is administered.

(C) Additional Considerations for Administration

Suitable animal subjects for administration of the compositionsdisclosed herein include, for example, fish, birds, cattle, pigs,horses, deer, sheep, goats, bison, rabbits, cats, dogs, chickens, ducks,turkeys, and the like. The mammal is preferably a human. Where thevaccine is for prophylactic use, the human is preferably a child (e.g.,a toddler or infant), a teenager, or an adult; where the vaccine is fortherapeutic use, the human is preferably a teenager or an adult. Avaccine intended for children may also be administered to adults, e.g.,to assess safety, dosage, immunogenicity, etc.

One way of checking efficacy of therapeutic treatment involvesmonitoring pathogen infection after administration of the compositionsor vaccines disclosed herein. One way of checking efficacy ofprophylactic treatment involves monitoring immune responses,systemically (such as monitoring the level of IgG1 and IgG2a production)and/or mucosally (such as monitoring the level of IgA production),against the antigen. Typically, antigen-specific serum antibodyresponses are determined post-immunization but pre-challenge whereasantigen-specific mucosal antibody responses are determinedpost-immunization and post-challenge.

Another way of assessing the immunogenicity of the compositions orvaccines disclosed herein where the nucleic acid molecule (e.g., theRNA) encodes a protein antigen is to express the protein antigenrecombinantly for screening patient sera or mucosal secretions byimmunoblot and/or microarrays. A positive reaction between the proteinand the patient sample indicates that the patient has mounted an immuneresponse to the protein in question. This method may also be used toidentify immunodominant antigens and/or epitopes within proteinantigens.

The efficacy of the compositions can also be determined in vivo bychallenging appropriate animal models of the pathogen of interestinfection.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

The compositions disclosed herein that include one or more antigens orare used in conjunction with one or more antigens may be used to treatboth children and adults. Thus a human subject may be less than 1 yearold, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55years old. Preferred subjects for receiving the compositions are theelderly (e.g., >50 years old, >60 years old, and preferably >65 years),the young (e.g., <5 years old), hospitalized patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The compositions are notsuitable solely for these groups, however, and may be used moregenerally in a population.

Preferred routes of administration include, but are not limited to,intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterial, and intraoccular injection. Oral and transdermaladministration, as well as administration by inhalation or suppositoryis also contemplated. Particularly preferred routes of administrationinclude intramuscular, intradermal and subcutaneous injection. Accordingto some embodiments of the present invention, the composition isadministered to a host animal using a needleless injection device, whichare well-known and widely available.

It is sometimes advantageous to employ a vaccine that targets aparticular target cell type (e.g., an antigen presenting cell or anantigen processing cell).

Catheters or like devices may be used to deliver the composition of theinvention, as polypeptide+naked RNA, polypeptide+RNA formulated with adelivery system (e.g., RNA encapsulated in liposomes), RNA only, orpolypeptide only into a target organ or tissue. Suitable catheters aredisclosed in, e.g., U.S. Pat. Nos. 4,186,745; 5,397,307; 5,547,472;5,674,192; and 6,129,705, all of which are incorporated herein byreference. The RNA molecules of the invention can also be introduceddirectly into a tissue, such as muscle. See, e.g., U.S. Pat. No.5,580,859. Other methods such as “biolistic” or particle-mediatedtransformation (see, e.g., Sanford et al., U.S. Pat. Nos. 4,945,050;5,036,006) are also suitable for introduction of RNA into cells of amammal. These methods are useful not only for in vivo introduction ofRNA into a mammal, but also for ex vivo modification of cells forreintroduction into a mammal.

The present invention includes the use of suitable delivery systems,such as liposomes, polymer microparticles or submicron emulsionmicroparticles with encapsulated or adsorbed RNA, or RNA+polypeptide, todeliver the RNA, or RNA+polypeptide, to elicit an immune response. Theinvention includes liposomes, microparticles, submicron emulsions, orcombinations thereof, with adsorbed and/or encapsulated RNA, orRNA+polypeptide.

The compositions disclosed herein that include one or more antigens, orare used in conjunction with one or more antigens, may be administeredto patients at substantially the same time as (e.g., during the samemedical consultation or visit to a healthcare professional orvaccination centre) other vaccines, e.g., at substantially the same timeas a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine,a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine, a DTP vaccine, a conjugated H. influenzaetype b vaccine, an inactivated poliovirus vaccine, a hepatitis B virusvaccine, a meningococcal conjugate vaccine (such as a tetravalent A CW135 Y vaccine), a respiratory syncytial virus vaccine, etc.

6. Definitions

The term “about”, as used here, refers to +/−10% of a value.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both), that elicits an immunologicalresponse.

An “epitope” is a portion of an antigen that is recognized by the immunesystem (e.g., by an antibody, an immunoglobulin receptor, a B cellreceptor, or a T cell receptor). An epitope can be linear orconformational. Commonly, an epitope is a polypeptide or polysaccharidein a naturally occurring antigen. In artificial antigens it can be a lowmolecular weight substance such as an arsanilic acid derivative.

T-cells and B-cells recognize antigens in different ways. T-cellsrecognize peptide fragments of proteins that are embedded in class-II orclass-I MHC molecules at the surface of cells, whereas B-cells recognizesurface features of an unprocessed antigen, via immunoglobulin-like cellsurface receptors. The difference in antigen recognition mechanisms ofT-cells and B-cells are reflected in the different natures of theirepitopes. Thus, whereas B-cells recognize surface features of an antigenor a pathogen, T-cell epitopes (which comprise peptides of about 8-12amino acids in length) can be “internal” as well as “surface” whenviewed in the context of the three-dimensional structure of the antigen.Accordingly, a B-cell epitope is preferably exposed on the surface ofthe antigen or pathogen, and can be linear or conformational, whereas aT-cell epitope is typically linear but is not required to be availableor on the surface of the antigen. Normally, a B-cell epitope willinclude at least about 5 amino acids but can be as small as 3-4 aminoacids. A T-cell epitope, such as a CTL epitope, will typically includeat least about 7-9 amino acids, and a helper T-cell epitope willtypically include at least about 12-20 amino acids.

When an individual is immunized with a polypeptide antigen havingmultiple epitopes, in many instances the majority of responding Tlymphocytes will be specific for one or a few linear epitopes from thatantigen and/or a majority of the responding B lymphocytes will bespecific for one or a few linear or conformational epitopes from thatantigen. Such epitopes are typically referred to as “immunodominantepitopes.” In an antigen having several immunodominant epitopes, asingle epitope may be most dominant, and is typically referred to as the“primary” immunodominant epitope. The remaining immunodominant epitopesare typically referred to as “secondary” immunodominant epitope(s).

The term “fusion polypeptide” refers to a single polypeptide in whichthe amino acid sequence is derived from at least two different naturallyoccurring proteins or polypeptide chains.

The term “naked” as used herein refers to nucleic acids that aresubstantially free of other macromolecules, such as lipids, polymers,and proteins. A “naked” nucleic acid, such as a self-replicating RNA, isnot formulated with other macromolecules to improve cellular uptake.Accordingly, a naked nucleic acid is not encapsulated in, absorbed on,or bound to a liposome, a microparticle or nanoparticle, a cationicemulsion, and the like.

As used herein, “nucleotide analog” or “modified nucleotide” refers to anucleotide that contains one or more chemical modifications (e.g.,substitutions) in or on the nitrogenous base of the nucleoside (e.g.,cytosine (C), thymine (T) or uracil (U)), adenine (A) or guanine (G)). Anucleotide analog can contain further chemical modifications in or onthe sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modifiedribose, modified deoxyribose, six-membered sugar analog, or open-chainsugar analog), or the phosphate.

The term “pathogen” refers to a virus, eukaryote, prokaryote or archaeathat is capable of proliferation, and causes a disease or illness in ahost organism, such as a vertebrate (e.g., a mammal). A pathogen can bea viral, bacterial, protozoan, or fungal species, as well as amulti-cellular parasitic species. As used herein, two epitopes are fromthe same pathogen when the two epitopes are from the same pathogenspecies, but not necessarily from the same strain, serotype, clade, etc.Therefore, the two epitopes can be from two different subspecies,strains, or serotypes of the same pathogen (e.g., one epitope from H1N1influenza virus, the other epitope from H3N2 influenza virus; oneepitope from HIV-1 Clade B, the other epitope from HIV-1 Clade C; etc.).

As used herein, a “polypeptide antigen” refers to a polypeptidecomprising one or more epitopes (either linear, conformational or both),that elicits an immunological response. Polypeptide antigens include,for example, a naturally-occurring protein, a mutational variant of anaturally-occurring protein (e.g., a protein that has amino acidsubstitution(s), addition(s), or deletion(s)), a truncated form of anaturally-occurring protein (e.g., an intracellular domain orextracellular domain of a membrane-anchored protein), as well as afusion protein (a protein that is derived from at least two differentnaturally occurring proteins or polypeptide chains). In addition,polypeptide antigens also encompass polypeptides that comprise one ormore amino acid stereoisomers, derivatives, or analogues. For example,amino acid derivatives include, e.g., chemical modifications of aminoacids such as alkylation, acylation, carbamylation, iodination, etc.Amino acid analogues include, e.g., compounds that have the same basicchemical structure as a naturally occurring amino acid, such ashomoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Polypeptide antigens also encompass polypeptides that aremodified post-translationally (such as acetylated, phosphorylated, orglycosylated polypeptides). Therefore, an epitope of a polypeptideantigen is not limited to a peptide. For example, an epitope of aglycosylated polypeptide may be a saccharide group that is attached tothe polypeptide chain.

Two protein antigens are “substantially the same” if the amino acidsequence identify between the two antigens is at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%, across the length of the shorter antigen.

The terms “treat,” “treating” or “treatment”, as used herein, includealleviating, abating or ameliorating disease or condition symptoms,preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition. The terms “treat,”“treating” or “treatment”, include, but are not limited to, prophylacticand/or therapeutic treatments

The term “viral replicon particle” or “VRP” refers to recombinantinfectious virions that cannot generate infectious progeny because ofdeletion of structural gene(s).

The term “virus-like particle” or “VLP” refers to a structure formed byviral coat proteins (e.g., a capsid) and optionally an evelope, buthaving no genetic material. A VLP resembles a viral particle.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Methods:

RNA Synthesis

Plasmid DNA encoding alphavirus replicons (see sequences, vA317, vA17,vA336, vA160, vA322, vA311, vA306, vA142, vA526, vA527, vA318, vA140,vA318, vA372, vA368, vA369) served as a template for synthesis of RNA invitro. Replicons contain the genetic elements required for RNAreplication but lack those encoding gene products necessary for particleassembly, the structural genes of the alphavirus genome are replaced bysequences encoding a heterologous protein. Upon delivery of thereplicons to eukaryotic cells, the positive-stranded RNA is translatedto produce four non-structural proteins, which together replicate thegenomic RNA and transcribe abundant subgenomic mRNAs encoding theheterologous gene product. Due to the lack of expression of thealphavirus structural proteins, replicons are incapable of inducing thegeneration of infectious particles. A bacteriophage (T7 or SP6) promoterupstream of the alphavirus cDNA facilitates the synthesis of thereplicon RNA in vitro and the hepatitis delta virus (HDV) ribozymeimmediately downstream of the poly(A)-tail generates the correct 3′-endthrough its self-cleaving activity.

Following linearization of the plasmid DNA downstream of the HDVribozyme with a suitable restriction endonuclease, run-off transcriptswere synthesized in vitro using T7 or SP6 bacteriophage derivedDNA-dependent RNA polymerase. Transcriptions were performed for 2 hoursat 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNApolymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP andUTP) following the instructions provided by the manufacturer (Ambion,Austin, Tex.). Following transcription, the template DNA was digestedwith TURBO™ DNASE enzyme (Ambion, Austin, Tex.). The replicon RNA wasprecipitated with LiCl and reconstituted in nuclease-free water.Uncapped RNA was capped post-transcripionally with Vaccinia CappingEnzyme (VCE) using the SCRIPTCAP™ m⁷G Capping System (EpicentreBiotechnologies, Madison, Wis.) as outlined in the user manual.Post-transcriptionally capped RNA was precipitated with LiCl andreconstituted in nuclease-free water. The concentration of the RNAsamples was determined by measuring the optical density at 260 nm.Integrity of the in vitro transcripts was confirmed by denaturingagarose gel electrophoresis.

LNP Formulation

1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DlinDMA) was synthesizedusing a previously published procedure [Heyes, J., Palmer, L., Bremner,K., MacLachlan, I. Cationic lipid saturation influences intracellulardelivery of encapsulated nucleic acids. Journal of Controlled Release,107: 276-287 (2005)]. 1, 2-Diastearoyl-sn-glycero-3-phosphocholine(DSPC) was purchased from Genzyme. Cholesterol was obtained fromSigma-Aldrich (St. Lois, Mo.). 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (PEG DMG 2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (PEG DMG 1000) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (PEG DMG 3000) were obtained from AvantiPolar Lipids (Alabaster, Ala.). 1,2-dioleoyl-3-trimethylammonium-propane(chloride salt) (DOTAP) and38-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride(DC-chol) were obtained from Avanti Polar Lipids. Proprietary lipidsRV03, RV04, RV05, RV06, RV07, RV08, RV09, RV10, RV11, RV12, RV15, weremade as previously described in WO 2011/076807, which disclosed thelipids and methods for making them. Lipid RV02 can be prepared accordingto known methods including the methods disclosed in WO 2011/057020. Forexample by substituting the alpha-amino and carbonyl of NorArgenine witha long chain alkenoyl and a long chain alkylamino, respectively. RV02has the structure

LNPs were formulated using three methods:

Method a (40 μg Batch, No Mustang, No Second Mixing, No TFF, withDialysis)

Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA,11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg of PEG DMG 2000 wereweighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipidstock solution was gently rocked at 37° C. for about 15 min to form ahomogenous mixture. Then, 120.9 μL of the stock was added to 1.879 mLethanol to make a working lipid stock solution of 2 mL. This amount oflipids was used to form LNPs with 40 μg RNA at a 8:1 N:P (Nitrogen toPhosphate) ratio. The protonatable nitrogen on DlinDMA (the cationiclipid) and phosphates on the RNA ae used for this calculation. Each μgof self-replicating RNA molecule was assumed to contain 3 nmoles ofanionic phosphate, each μg of DlinDMA was assumed to contains 1.6 nmolesof cationic nitrogen. A 2 mL working solution of RNA was also preparedfrom a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6)(Teknova). Three 20 mL glass vials (with stir bars) were rinsed withRNASE AWAY® solution (Molecular BioProducts) and washed with plenty ofMILLIQ® water before use to decontaminate the vials of RNAses. One ofthe vials was used for the RNA working solution and the others forcollecting the lipid and RNA mixes (as described later). The workinglipid and RNA solutions were heated at 37° C. for 10 min before beingloaded into 3 cc luer-lok syringes (BD Medical). 2 mL of citrate buffer(pH 6) was loaded in another 3 cc syringe. Syringes containing RNA andthe lipids were connected to a T mixer (PEEK™ 500 μm ID junction) usingFEP tubing ([fluorinated ethylene-propylene] 2 mm ID×3 mm OD, IdexHealth Science, Oak Harbor, Wash.). The outlet from the T mixer was alsoFEP tubing (2 mm ID×3 mm). The third syringe containing the citratebuffer was connected to a separate piece of tubing (2 mm ID×3 mm OD).All syringes were then driven at a flow rate of 7 mL/min using a syringepump (from kdScientific, model no. KDS-220). The tube outlets werepositioned to collect the mixtures in a 20 mL glass vial (whilestirring). Next, LNPs were loaded into Pierce SLIDE-A-LYZER™ DialysisCassettes (Thermo Scientific, extra strength, 0.5-3 mL capacity) anddialyzed against 400-500 mL of 1×PBS (diluted from 10× ACCUGENE® PBS,from Lonza) overnight at 4° C. in an autoclaved plastic container beforerecovering the final product. For in vitro and in vivo experiments,formulations were diluted to the required RNA concentration with 1×PBS(from Teknova).

pKas

Unless explicitly indicated otherwise, all pKas referred to herein aremeasured in water at standard temperature and pressure. Also, unlessotherwise indicated, all references to pKa are references to pKameasured using the following technique. 2 mM solution of lipid inethanol are prepared by weighing the lipid and then dissolving inethanol. 0.3 mM solution of fluorescent probe TNS in ethanol:methanol9:1 is prepared by first making 3 mM solution of TNS in methanol andthen diluting to 0.3 mM with ethanol.

An aqueous buffer containing sodium phosphate, sodium citrate, sodiumacetate and sodium chloride, at the concentrations 20 mM, 25 mM, 20 mMand 150 mM, respectively, is prepared. The buffer is split into eightparts and the pH adjusted either with 12N HCl or 6N NaOH to 4.44-4.52,5.27, 6.15-6.21, 6.57, 7.10-7.20, 7.72-7.80, 8.27-8.33 and 10.47-11.12.400 uL of 2 mM lipid solution and 800 uL of 0.3 mM TNS solution aremixed.

Using the Tecan Genesis RSP150 high throughput liquid handler and GeminiSoftware, 7.5 uL of probe/lipid mix are added to 242.5 uL of buffer in a1 mL 96 well plate (model NUNC 260252, Nalgae Nunc International). Thisis done with all eight buffers.

After mixing in 1 mL 96 well plate, 100 uL of each probe/lipid/buffermixture is transferred to a 250 uL black with clear bottom 96 well plate(model COSTAR 3904, Corning). The fluorescence measurements are carriedout on the SPECTRAMAX® M5 spectrophotometer using software SoftMax pro5.2 and following parameters:

Read Mode: Fluorescence, Top read

Wavelengths: Ex 322 nm, Em 431 nm, Auto Cutoff On 420 nm Sensitivity:Readings 6, PMT: Auto Automix: Before: Off Autocalibrate: On

Assay plate type: 96 Well Standard clrbtmWells to read: Read entire plateSettling time: OffColumn Wav. Priority: Column priority

Carriage Speed: Normal

Auto read: Off

After the measurement, the background fluorescence value of an emptywell on the 96 well plate is subtracted from each probe/lipid/buffermixture. The fluorescence intensity values are then normalized to thevalue at lowest pH. The normalized fluorescence intensity vs. pH chartis then plotted in the Microsoft Excel software. The eight points areconnected with a smooth line.

The point on the line at which the normalized fluorescence intensity isequal to 0.5 is found. The pH corresponding to normalized fluorescenceintensity equal to 0.5 is found and is considered the pKa of the lipid.

Method B (75 μg Batch, PES Hollow Fibers and No Mustang):

Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA,11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg of PEG DMG 2000 wereweighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipidstock solution was gently rocked at 37° C. for about 15 min to form ahomogenous mixture. Then, 226.7 μL of the stock was added to 1.773 mLethanol to make a working lipid stock solution of 2 mL. This amount oflipids was used to form LNPs with 75 μg RNA at a 8:1 N:P (Nitrogen toPhosphate) ratio. The protonatable nitrogen on DlinDMA (the cationiclipid) and phosphates on the RNA are used for this calculation. Each μgof self-replicating RNA molecule was assumed to contain 3 nmoles ofanionic phosphate, each μg of DlinDMA was assumed to contains 1.6 nmolesof cationic nitrogen. A 2 mL working solution of RNA was also preparedfrom a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6)(Teknova). Three 20 mL glass vials (with stir bars) were rinsed withRNASE AWAY® solution (Molecular BioProducts) and washed with plenty ofMILLIQ® water before use to decontaminate the vials of RNAses. One ofthe vials was used for the RNA working solution and the others forcollecting the lipid and RNA mixes (as described later). The workinglipid and RNA solutions were heated at 37° C. for 10 min before beingloaded into 3 cc luer-lok syringes (BD Medical). 2 mL of citrate buffer(pH 6) was loaded in another 3 cc syringe. Syringes containing RNA andthe lipids were connected to a T mixer (PEEK™ 500 μm ID junction) usingFEP tubing ([fluorinated ethylene-propylene] 2 mm ID×3 mm OD, IdexHealth Science, Oak Harbor, Wash.). The outlet from the T mixer was alsoFEP tubing (2 mm ID×3 mm). The third syringe containing the citratebuffer was connected to a separate piece of tubing (2 mm ID×3 mm OD).All syringes were then driven at a flow rate of 7 mL/min using a syringepump (from kdScientific, model no. KDS-220). The tube outlets werepositioned to collect the mixtures in a 20 mL glass vial (whilestirring). The stir bar was taken out and the ethanol/aqueous solutionwas allowed to equilibrate to room temperature for 1 h. Then the mixturewas loaded in a 5 cc syringe (BD Medical), which was fitted to a pieceof FEP tubing (2 mm ID×3 mm OD) and in another 5 cc syringe with equallength of FEP tubing, an equal volume of 100 mM citrate buffer (pH 6)was loaded. The two syringes were driven at 7 mL/min flow rate using asyringe pump and the final mixture collected in a 20 mL glass vial(while stirring). Next, LNPs were concentrated to 2 mL and dialyzedagainst 10-15 volumes of 1×PBS (from Teknova) using the Tangential FlowFiltration (TFF) system before recovering the final product. The TFFsystem and hollow fiber filtration membranes were purchased fromSpectrum Labs and were used according to the manufacturer's guidelines.Polyethersulfone (PES) hollow fiber filtration membranes (part numberP-C1-100E-100-01N) with a 100 kD pore size cutoff and 20 cm² surfacearea were used. For in vitro and in vivo experiments, formulations werediluted to the required RNA concentration with 1×PBS (from Teknova).

Method C (75 μg Batch, with Mustang and PES Hollow Fibers):

Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA,11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg of PEG DMG 2000 wereweighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipidstock solution was gently rocked at 37° C. for about 15 min to form ahomogenous mixture. Then, 226.7 μL of the stock was added to 1.773 mLethanol to make a working lipid stock solution of 2 mL. This amount oflipids was used to form LNPs with 75 μg RNA at a 8:1 N:P (Nitrogen toPhosphate) ratio. The protonatable nitrogen on DlinDMA (the cationiclipid) and phosphates on the RNA are used for this calculation. Each μgof self-replicating RNA molecule was assumed to contain 3 nmoles ofanionic phosphate, each μg of DlinDMA was assumed to contains 1.6 nmolesof cationic nitrogen. A 2 mL working solution of RNA was also preparedfrom a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6)(Teknova). Three 20 mL glass vials (with stir bars) were rinsed withRNASE AWAY® solution (Molecular BioProducts) and washed with plenty ofMILLIQ® water before use to decontaminate the vials of RNAses. One ofthe vials was used for the RNA working solution and the others forcollecting the lipid and RNA mixes (as described later). The workinglipid and RNA solutions were heated at 37° C. for 10 min before beingloaded into 3 cc luer-lok syringes (BD Medical). 2 mL of citrate buffer(pH 6) was loaded in another 3 cc syringe. Syringes containing RNA andthe lipids were connected to a T mixer (PEEK™ 500 μm ID junction) usingFEP tubing ([fluorinated ethylene-propylene] 2 mm ID×3 mm OD, IdexHealth Science, Oak Harbor, Wash.). The outlet from the T mixer was alsoFEP tubing (2 mm ID×3 mm). The third syringe containing the citratebuffer was connected to a separate piece of tubing (2 mm ID×3 mm OD).All syringes were then driven at a flow rate of 7 mL/min using a syringepump (from kdScientific, model no. KDS-220). The tube outlets werepositioned to collect the mixtures in a 20 mL glass vial (whilestirring). The stir bar was taken out and the ethanol/aqueous solutionwas allowed to equilibrate to room temperature for 1 h. Then the mixturewas loaded in a 5 cc syringe (BD Medical), which was fitted to a pieceof FEP tubing (2 mm ID×3 mm OD) and in another 5 cc syringe with equallength of FEP tubing, an equal volume of 100 mM citrate buffer (pH 6)was loaded. The two syringes were driven at 7 mL/min flow rate using asyringe pump and the final mixture collected in a 20 mL glass vial(while stirring). Next, the mixture collected from the second mixingstep (LNPs) were passed through MUSTANG® Q membrane (an anion-exchangesupport that binds and removes anionic molecules, obtained from PallCorporation, AnnArbor, Mich., USA). Before passing the LNPs, 4 mL of 1 MNaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6) weresuccessively passed through the Mustang membrane. LNPs were warmed for10 min at 37° C. before passing through the mustang filter. Next, LNPswere concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS(from Teknova) using the Tangential Flow Filtration (TFF) system beforerecovering the final product. The TFF system and hollow fiber filtrationmembranes were purchased from Spectrum Labs and were used according tothe manufacturer's guidelines. Polyethersulfone (PES) hollow fiberfiltration membranes (part number P-C1-100E-100-01N) with a 100 kD poresize cutoff and 20 cm² surface area were used. For in vitro and in vivoexperiments, formulations were diluted to the required RNA concentrationwith 1×PBS (from Teknova).

CNE Formulations

CNEs were prepared similar to charged MF59 as previously described (Ottet al., Journal of Controlled Release, volume 79, pages 1-5, 2002), withone major modification for CMF34. DOTAP was dissolved in the squalenedirectly, and no organic solvent was used. It was discovered thatinclusion of a solvent in emulsions that contained greater than 1.6mg/ml DOTAP produced a foamy feedstock that could not be microfluidizedto produce an emulsion. Heating squalene to 37° C. allowed DOTAP to bedirectly dissolved in squalene, and then the oil phase could besuccessfully dispersed in the aqueous phase (e.g., by homogenization) toproduce an emulsion.

TABLE 3 Cationic oil:Lipid Lipid ratio Aqueous CNE mg/mL SurfactantSqualene (mole:mole) phase CNE13 DDA 0.5% SPAN ® 85 4.3% 10 mM DDA (inDCM) 0.5% TWEEN ® 80 citrate (in DCM) 1.45 buffer pH 6.5 CNE17 DOTAP0.5% SPAN ® 85 4.3% 52.4:1 10 mM (in DCM) 0.5% TWEEN ® 80 citrate 1.4buffer pH 6.5 CMF34 DOTAP 0.5% SPAN ® 85 4.3% 16.7:1 10 mM (no organic0.5% TWEEN ® 80 citrate solvent) buffer 4.4 pH 6.5

RNA Complexation

The number of nitrogens in solution was calculated from the cationiclipid concentration, DOTAP for example has 1 nitrogen that can beprotonated per molecule. The RNA concentration was used to calculate theamount of phosphate in solution using an estimate of 3 nmols ofphosphate per microgram of RNA. By varying the amount of RNA:Lipid, theN/P ratio can be modified. RNA was complexed to the CNEs in a range ofnitrogen/phosphate ratios (N/P). Calculation of the N/P ratio was doneby calculating the number of moles of protonatable nitrogens in theemulsion per milliliter. To calculate the number of phosphates, aconstant of 3 nmols of phosphate per microgram of RNA was used.

After the values were determined, the appropriate ratio of the emulsionwas added to the RNA. Using these values, the RNA was diluted to theappropriate concentration and added directly into an equal volume ofemulsion while vortexing lightly. The solution was allowed to sit atroom temperature for approximately 2 hours. Once complexed the resultingsolution was diluted to the appropriate concentration and used within 1hour.

Particle Size

Particle size was measured using a Zetasizer Nano ZS zeta potentialanalyzer (Malvern Instruments, Worcestershire, UK) according to themanufacturer's instructions. Particle sizes are reported as the Zaverage with the polydispersity index (pdi). Liposomes were diluted in1×PBS before measurement.

Encapsulation Efficiency and RNA Concentration

The percentage of encapsulated RNA and RNA concentration were determinedby QUANT-IT RIBOGREEN® RNA reagent kit (Invitrogen). Manufacturer'sinstructions were followed in the assay. The ribosomal RNA standardprovided in the kit was used to generate a standard curve. LNPs werediluted ten fold or one hundred fold in 1×TE buffer (from kit), beforeaddition of the dye. Separately, LNPs were diluted ten or 100 fold in1×TE buffer containing 0.5% TRITON™ X surfactant (Sigma-Aldrich), beforeaddition of the dye. Thereafter an equal amount of dye was added to eachsolution and then ˜180 μL of each solution after dye addition was loadedin duplicate into a 96 well tissue culture plate (obtained from VWR,catalog #353072). The fluorescence (Ex 485 nm, Em 528 nm) was read on amicroplate reader (from BioTek Instruments, Inc.).

TRITON™ X surfactant was used to disrupt the LNPs, providing afluorescence reading corresponding to the total RNA amount and thesample without TRITON™ X surfactant provided fluorescence correspondingto the unencapsulated RNA. % RNA encapsulation was determined asfollows: LNP RNA Encapsulation (%)=[(F_(t)−F_(i))/F_(t)]×100, whereF_(t) is the fluorescence intensity of LNPs with TRITON™ X surfactantaddition and F_(i) is the fluorescence intensity of the LNP solutionwithout detergent addition. These values (F_(t) and F_(i)) were obtainedafter subtraction from blank (1×TE buffer) fluorescence intensity. Theconcentration of encapsulated RNA was obtained by comparing F_(t)−F_(i)with the standard curve generated. All LNP formulations were dosed invivo based on the encapsulated dose.

Gel Electrophoresis

Denaturing gel electrophoresis was performed to evaluate the integrityof the RNA after the formulation process and to assess the RNAseprotection of the encapsulated RNA. The gel was cast as follows: 0.4 gof agarose (Bio-Rad, Hercules, Calif.) was added to 36 ml of DEPCtreated water and heated in a microwave until dissolved and then cooleduntil warm. 4 ml of 10× denaturing gel buffer (Ambion, Austin, Tex.),was then added to the agarose solution. The gel was poured and wasallowed to set for at least 30 minutes at room temperature. The gel wasthen placed in a gel tank, and 1× NORTHERNMAX® running buffer (Ambion,Austin, Tex.) was added to cover the gel by a few millimeters.

RNase Protection Assay

RNase digestion was achieved by incubation with 3.8 mAU of RNase A permicrogram of RNA (Ambion, Hercules, and CA) for 30 minutes at roomtemperature. RNase was inactivated with Protenase K (Novagen, Darmstadt,Germany) by incubating the sample at 55° C. for 10 minutes. Post RNaseinactivation, a 1:1 v/v mixture of sample to 25:24:1 v/v/v,phenol:chloroform:isoamyl alcohol was added to extract the RNA from thelipids into the aqueous phase. Samples were mixed by vortexing for a fewseconds and then placed on a centrifuge for 15 minutes at 12 k RPM. Theaqueous phase (containing the RNA) was removed and used to analyze theRNA. Prior to loading (400 ng RNA per well) all the samples wereincubated with formaldehyde loading dye, denatured for 10 minutes at 65°C. and cooled to room temperature. Ambion MILLENNIUM™ markers were usedto approximate the molecular weight of the RNA construct. The gel wasrun at 90 V. The gel was stained using 0.1% SYBR gold according to themanufacturer's guidelines (Invitrogen, Carlsbad, Calif.) in water byrocking at room temperature for 1 hour. Gel images were taken on aBio-Rad CHEMIDOC™ XRS imaging system (Hercules, Calif.).

Secreted Alkaline Phosphatase (SEAP) Assay

To assess the kinetics and amount of antigen production in vivo, an RNAreplicon encoding for SEAP was administered with and without formulationto mice via intramuscularly injection. Groups of 5 female BALB/c miceaged 8-10 weeks and weighing about 20 g were immunized with liposomesencapsulating RNA encoding for SEAP. Naked RNA was administered in RNasefree 1×PBS. As a positive control, viral replicon particles (VRPs) at adose of 5×10⁵ infectious units (IU) were also sometimes administered. A100 μl dose was administered to each mouse (50 μl per site) in thequadriceps muscle. Blood samples were taken 1, 3, and 6 days postinjection. Serum was separated from the blood immediately aftercollection, and stored at −30° C. until use.

A chemiluminescent SEAP assay PHOSPHA-LIGHT™ System (Applied Biosystems,Bedford, Mass.) was used to analyze the serum. Mouse sera were diluted1:4 in 1× PHOSPHA-LIGHT™ dilution buffer. Samples were placed in a waterbath sealed with aluminum sealing foil and heat inactivated for 30minutes at 65° C. After cooling on ice for 3 minutes, and equilibratingto room temperature, 50 μL of PHOSPHA-LIGHT™ assay buffer was added tothe wells and the samples were left at room temperature for 5 minutes.Then, 50 μL of reaction buffer containing 1:20 CSPD® (chemiluminescentalkaline phosphate substrate) substrate was added, and the luminescencewas measured after 20 minutes of incubation at room temperature.Luminescence was measured on a Berthold Centro LB 960 luminometer (OakRidge, Tenn.) with a 1 second integration per well. The activity of SEAPin each sample was measured in duplicate and the mean of these twomeasurements taken.

Viral Replicon Particles (VRP)

To compare RNA vaccines to traditional RNA-vectored approaches forachieving in vivo expression of reporter genes or antigens, we utilizedviral replicon particles (VRPs) produced in BHK cells by the methodsdescribed by Perri et al. (2003) An alphavirus replicon particle chimeraderived from venezuelan equine encephalitis and sindbis viruses is apotent gene-based vaccine delivery vector. J Virol 77: 10394-10403. Inthis system, the antigen (or reporter gene) replicons consisted ofalphavirus chimeric replicons (VCR) derived from the genome ofVenezuelan equine encephalitis virus (VEEV) engineered to contain the 3′terminal sequences (3′ UTR) of Sindbis virus and a Sindbis viruspackaging signal (PS) (see FIG. 2 of Perri et al). These replicons werepackaged into VRPs by co-electroporating them into baby hamster kidney(BHK) cells along with defective helper RNAs encoding the Sindbis viruscapsid and glycoprotein genes (see FIG. 2 of Perri et al., J. Virol. 77:10394-10403 (2003)). The VRPs were then harvested and titrated bystandard methods and inoculated into animals in culture fluid or otherisotonic buffers.

RSV-F Trimer Subunit Vaccine

The RSV F trimer is a recombinant protein comprising the ectodomain ofRSV F with a deletion of the fusion peptide region preventingassociation with other trimers. The resulting construct forms ahomogeneous trimer, as observed by size exclusion chromatography, andhas an expected phenotype consistent with a postfusion F conformation asobserved by electron microscopy. The protein was expressed in insectcells and purified by virtue of a HIS-tagged in fusion with theconstruct's C-terminus followed by size exclusion chromatography usingconventional techniques. The resulting protein sample exhibits greaterthan 95% purity. For the in vivo evaluation of the F-subunit vaccine,100 μg/mL trimer protein was adsorbed on 2 mg/mL alum using 10 mMHistidine buffer, pH 6.3 and isotonicity adjusted with sodium chlorideto 150 mM. F-subunit protein was adsorbed on alum overnight with gentlestirring at 2-8° C.

Murine Immunogenicity Studies

Groups of 10 female BALB/c mice aged 8-10 weeks and weighing about 20 gwere immunized at day 0 and day 21 with bleeds taken at days 14, 35 and49. All animals were injected in the quadriceps in the two hind legseach getting an equivalent volume (50 μl per site). When measurement ofT cell responses was required, spleens were harvested at day 35 or 49.

Vaccination and Challenge of Cotton Rats

Female cotton rats (Sigmodon hispidis) were obtained from HarlanLaboratories. All experiments were approved and performed according toNovartis Animal Care and Use Committee. Groups of animals were immunizedintramuscularly (i.m., 100 μl) with the indicated vaccines on days 0 and21. Serum samples were collected 2 weeks after each immunization.Immunized or unvaccinated control animals were challenged intranasally(i.n.) with 1×10⁵ PFU RSV 4 weeks after the final immunization. Bloodcollection and RSV challenge were performed under anesthesia with 3%isoflurane using a precision vaporizer.

RSV F-Specific ELISA

Individual serum samples were assayed for the presence of RSV F-specificIgG by enzyme-linked immunosorbent assay (ELISA). ELISA plates (MaxiSorp96-well, Nunc) were coated overnight at 4° C. with 1 g/ml purified RSV Fin PBS. After washing (PBS with 0.1% TWEEN®-20 surfactant), plates wereblocked with Superblock Blocking Buffer in PBS (Thermo Scientific) forat least 1.5 hr at 37° C. The plates were then washed, serial dilutionsof serum in assay diluent (PBS with 0.1% TWEEN®-20 surfactant and 5%goat serum) from experimental or control cotton rats were added, andplates were incubated for 2 hr at 37° C. After washing, plates wereincubated with horse radish peroxidase (HRP)-conjugated chickenanti-cotton rat IgG (Immunology Consultants Laboratory, Inc, diluted1:5,000 in assay diluent) for 1 hr at 37° C. Finally, plates were washedand 100 μl of TMB peroxidase substrate solution (Kirkegaard & PerryLaboratories, Inc) was added to each well. Reactions were stopped byaddition of 100 μl of 1M H₃PO₄, and absorbance was read at 450 nm usinga plate reader. For each serum sample, a plot of optical density (OD)versus logarithm of the reciprocal serum dilution was generated bynonlinear regression (GraphPad Prism). Titers were defined as thereciprocal serum dilution at an OD of approximately 0.5 (normalized tostandard, pooled sera from RSV-infected cotton rats with a defined titerof 1:2500, that was included on every plate).

RSV Micro Neutralization Assay

Serum samples were tested for the presence of neutralizing antibodies bya plaque reduction neutralization test (PRNT). Two-fold serial dilutionsof HI-serum (in PBS with 5% HI-FBS) were added to an equal volume of RSVLong previously titered to give approximately 115 PFU/25 μl. Serum/virusmixtures were incubated for 2 hours at 37° C. and 5% CO2, to allow virusneutralization to occur, and then 25 μl of this mixture (containingapproximately 115 PFU) was inoculated on duplicate wells of HEp-2 cellsin 96 well plates. After 2 hr at 37° C. and 5% CO2, the cells wereoverlayed with 0.75% Methyl Cellulose/EMEM 5% HI-FBS and incubated for42 hours. The number of infectious virus particles was determined bydetection of syncytia formation by immunostaining followed by automatedcounting. The neutralization titer is defined as the reciprocal of theserum dilution producing at least a 60% reduction in number of synctiaper well, relative to controls (no serum).

CMV Micro Neutralization Assay

Serum samples were tested for the presence of neutralizing antibodies byan infection reduction neutralization test. Two-fold serial dilutions ofHI-serum (in DMEM with 10% HI FBS) were added to an equal volume of CMV(strain TB40 or clinical isolate 8819) previously titered to giveapproximately 200 IU/50 μl. Serum/virus mixtures were incubated for 2hours at 37° C. and 5% CO2, to allow virus neutralization to occur, andthen 50 μl of this mixture (containing approximately 200 IU) wasinoculated on duplicate wells of ARPE-19 cells in 96 half well plates.Plates were incubated for 40-44 hours. The number of positive infectedfoci was determined by immunostaining with an AlexaFluor 488 conjugatedIE1 CMV monoclonal antibody followed by automated counting. Theneutralization titer is defined as the reciprocal of the serum dilutionproducing a 50% reduction in number of positive virus foci per well,relative to controls (no serum).

Bovine Immunogenicity Studies

Twenty colostrum deprived Holstein/Holstein cross calves were used inthe study. All calves were male. Animals were obtained from J & R, alocal supplier and double ear-tagged for identification, as per siteprocedures. Initially, there was no intent to castrate and dehorn,however, when the study was extended to include 3^(rd) and 4^(th)vaccinations, these procedures were performed to help ensure animal andhandler safety. The procedures were performed in accordance with RACSOPs. The procedures were done at a time felt to have the least impacton the study results. All calves were prescreened prior to the beginningof the study, and were seronegative for BRSV via a serum neutralizationassay. Seronegative calves were defined as those having titers of ≤1:4titer to BRSV in a constant virus, decreasing serum neutralization (SN)assay with 50-500 Tissue Culture Infective Dose₅₀ (TCID₅₀) BRSV. Calveswere also screened prior to acquisition, for persistent infection withBovine Virus Diarrhea Virus (BVDV). Immunohistochemistry (IHC) testingof ear notch samples was performed with all negative results.

Calves (5 per group) were given intramuscular vaccinations on days 0,21, 86 and 146. RNA and PBS vaccines were administered as 1.0 mL splitdoses on each side of the neck (2 mL total dose). The Triangle 4 productwas administered as labeled (2.0 mL dose on one side of the neck). TheRSV-F subunit protein vaccine (15 μg) adjuvanted with MF59 wasadministered as 1.0 mL split doses on each side of the neck (2 mL totaldose). Serum was collected for antibody analysis on days 0, 14, 21, 35,42, 56, 63, 86, 100, 107, 114, 121, 128, 135, 146, 160, 167, 174, 181,188, 195, and 202.

Example I—Co-Administration of RNA and Protein to Mice

In this example, an RNA molecule encoding the RSV-F, or an RNA moleculeencoding the GFP protein, was co-administered with an RSV-F antigen inpolypeptide form. The effects of RNA on its “cognate” antigen and“non-cognate” antigen were assessed. The RSV-F antigen is a“non-cognate” antigen of the GFP-coding RNA because the F antigen doesnot share sequence homology to, and does not immunologically cross-reactwith the polypeptide encoded by the RNA molecule (GFP).

Three RNAs were used for this study: the vA317 replicon that expressesthe surface fusion glycoprotein of RSV (RSV-F); the vA17 replicon thatexpresses green fluorescent protein (GFP); and the vA336 replicon thatis replication-defective and encodes GFP. BALB/c mice, 5 animals pergroup, were given bilateral intramuscular vaccinations (50 μL per leg)on days 0 and 21. Spleens were harvested at day 49 for T cell analysis.Animals, 70 total, were divided into 14 groups (5 animals per group):

Group 1 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with VRPs (1×10⁶ IU) expressing the full-length wild typesurface fusion glycoprotein of RSV.

Group 2 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with the RSV-F subunit protein (5 μg).

Group 3 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with the RSV-F subunit protein vaccine (5 μg) adjuvantedwith alum and a small molecule TLR7 agonist (TLR7A, 25 μg).

Group 4 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed withself-replicating RNA (vA317, 1 μg, RSV-F) which had been formulated withCNE17 (prior to protein addition at an N:P ratio of 10:1).

Group 5 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed withself-replicating RNA (vA317, 1 μg, RSV-F) which had been formulated withLNPs (RV01(36)). The LNP had the following composition: 40% DlinDMA, 10%DSPC, 48% Chol, 2% PEG DMG 2000 and an N:P ratio of 8:1. They were madeusing Method A, except a 150 μg RNA batch size was used.

Group 6 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed with empty LNPs(RV01(36)), made as per group 5). The lipid dose was equivalent to thedose of the LNP formulation in group 9 and 12 which contained 0.01 μgRNA.

Group 7 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed with empty LNPs(RV01(36)), made as per group 5). The lipid dose was equivalent to thedose of the LNP formulation in group 10 and 13 which contained 0.1 μgRNA.

Group 8 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed with empty LNPs(RV01(36)), made as per group 5). The lipid dose was equivalent to thedose of the LNP formulation in group 11 and 14 which contained 1.0 μgRNA.

Group 9 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed withself-replicating RNA (vA17, 1 μg, GFP) formulated with LNPs (RV01(36)),made as per group 5). The lipid dose was equivalent to the dose of theLNP formulation in groups 6 and 12.

Group 10 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed withself-replicating RNA (vA17, 1 μg, GFP) formulated with LNPs (RV01(36)),made as per group 5). The lipid dose was equivalent to the dose of theLNP formulation in groups 7 and 13.

Group 11 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed withself-replicating RNA (vA17, 1 μg, GFP) formulated with LNPs (RV01(36),made as per group 5). The lipid dose was equivalent to the dose of theLNP formulation in groups 8 and 14.

Group 12 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed withnon-replicating RNA (vA336, 1 μg, GFP) formulated with LNPs (RV01(36)),made as per group 5). The lipid dose was equivalent to the dose of theLNP formulation in groups 6 and 9.

Group 13 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed withnon-replicating RNA (vA317, 1 μg, GFP) formulated with LNPs (RV01(36),made as per group 5). The lipid dose was equivalent to the dose of theLNP formulation in groups 7 and 10.

Group 14 were given bilateral intramuscular vaccinations (50 μL per leg)on days 0, 21 with RSV-F subunit protein (5 μg) mixed withnon-replicating RNA (vA317, 1 μg, GFP) formulated with LNPs (RV01(36),made as per group 5). The lipid dose was equivalent to the dose of theLNP formulation in groups 8 and 11.

Results and Conclusions

F-specific serum IgG titers are shown in tables I-1&2 (day 14) and I-3&4(day 35). F-specific serum IgG1 titers are shown in tables I-5&6 (day35) and F-specific serum IgG2a titers are shown in tables I-7-I-9 (day35). RSV serum neutralizing antibody titers are shown in table I-10(days 35 and 49). Average net F-specific cytokine-positive T cellfrequencies (CD4+ or CD8+) are shown in Tables I-11 and I-12.

This study showed that RNA formulated with LNP was a novel, potentadjuvant for recombinant protein (RSV F). LNP was tested without andwith RNA (encoding GFP) as adjuvants for F protein. Serum antibodies andsplenic T cells specific for F antigen were measured. The LNP RV01(without RNA) was an adjuvant for F protein. RV01 formulated with GFPRNA was even more effective, indicating that both the LNP and the RNAcomponents contribute to the adjuvanticity. RNA was needed to induceIgG2a, but not IgG1, indicating that LNP was not a “Th1” type adjuvant.

For the combination of protein (RSV F) and LNP/RNA, F RNA was comparedto GFP RNA and to no RNA (LNP present in all). For antibody responses,there was relatively little difference between F RNA, GFP RNA, and noRNA, except for IgG2a, as noted above. However, F RNA induced superiorCD8 T cell responses compared to GFP RNA or no RNA

TABLE I-1 F-specific serum IgG titers of BALB/c mice, 5 animals pergroup, 14 days after intramuscular vaccination. Data are represented astiters for individual mice and the geometric mean titers of 5 individualmice per group. If an individual animal had a titer of <25 (limit ofdetection), it was assigned a titer of 5. 1E6 IU 5 μg RSV F/ 5 μg RSVF + 5 μg RSV F + dose/ VRP-VCR2.1- 25 μg TLR7A/ 1 μg vA317u/ 1 μgvA317u/ vaccine RSV-F-full 5 μg RSV F alum CNE17 RV01(36) F- 1775 520850 1326 5464 specific 1976 5 13877 1816 14434 serum 3080 444 142392184 7587 IgG 1964 44 14590 1016 9180 titers 2003 5 19155 614 10938 GMT2117 19 16302 1268 9031

TABLE I-2 Continued from Table I-1. F-specific serum IgG titers ofBALB/c mice, 5 animals per group, 14 days after intramuscularvaccination. Data are represented as titers for individual mice and thegeometric mean titers of 5 individual mice per group. If an individualanimal had a titer of <25 (limit of detection), it was assigned a titerof 5. dose/vaccine 5 μg 5 μg 5 μg 5 μg 5 μg 5 μg 5 μg 5 μg 5 μg RSV F +RSV F + RSV F + RSV F + RSV F + RSV F + RSV F + RSV F + RSV F + RV01(36)RV01(36) RV01(36) 0.01 μg 0.1 μg 1 μg 0.01 μg 0.1 μg 1 μg empty emptyempty vA17u/ vA17u/ vA17u/ vA336u/ vA336u/ vA336u/ (0.01 μg) (0.1 μg) (1μg) RV01(36) RV01(36) RV01(36) RV01(36) RV01(36) RV01(36) F- 5 5 1949 5411 3412 5 478 1724 specific 5 53 4742 5 481 1835 37 641 3663 serum 5 372673 5 2727 1904 5 1115 4119 IgG 5 65 4133 218 447 2148 32 826 2759titers 5 129 783 317 1207 2106 5 1043 2640 GMT 5 38 2402 24 781 2220 11783 2854

TABLE I-3 F-specific serum IgG titers of BALB/c mice, 5 animals pergroup, intramuscular vaccinations on days 0 and 21. Serum was collectedfor antibody analysis on day 35 (2wp2). Data are represented as titersfor individual mice and the geometric mean titers of 5 individual miceper group. 1E6 IU 5 μg RSV F/ 5 μg RSV F + 5 μg RSV F + dose/VRP-VCR2.1- 25 μg TLR7A/ 1 μg vA317u/ 1 μg vA317u/ vaccine RSV-F-full 5μg RSV F Alum CNE17 RV01(36) F- 34973 1712 256096 106631 137187 specific27377 1528 233285 110416 195548 serum 46627 9472 372299 193437 177449IgG 47593 10788 200553 65757 225507 titers 17089 7053 246051 106100169730 GMT 32509 4519 255910 109704 178695

TABLE I-4 Continued from Table I-3. F-specific serum IgG titers ofBALB/c mice, 5 animals per group, intramuscular vaccinations on days 0and 21. Serum was collected for antibody analysis on day 35 (2wp2). Dataare represented as titers for individual mice and the geometric meantiters of 5 individual mice per group. dose/vaccine 5 μg 5 μg 5 μg 5 μg5 μg 5 μg 5 μg 5 μg 5 μg RSV F + RSV F + RSV F + RSV F + RSV F + RSV F +RSV F + RSV F + RSV F + RV01(36) RV01(36) RV01(36) 0.01 μg 0.1 μg 1 μg0.01 μg 0.1 μg 1 μg empty empty empty vA17u/ vA17u/ vA17u/ vA336u/vA336u/ vA336u/ (0.01 μg) (0.1 μg) (1 μg) RV01(36) RV01(36) RV01(36)RV01(36) RV01(36) RV01(36) F- 1916 5300 148066 10130 49413 190336 555978946 182029 specific 462 56079 213010 18791 49915 215482 39933 93558167670 serum 1197 7851 125433 19122 78043 199401 1694 77531 296722 IgG67 27879 160137 39695 131798 186112 6422 117371 225668 titers 2746 28847227934 66920 94756 287935 2973 155149 208995 GMT 721 17975 170574 2495075194 212983 5905 100841 211890

TABLE I-5 F-specific serum IgG1 titers of BALB/c mice, 5 animals pergroup, intramuscular vaccinations on days 0 and 21. Serum was collectedfor antibody analysis on day 35 (2wp2). Data are represented as titersfor individual mice and the geometric mean titers of 5 individual miceper group. 1E6 IU 5 μg RSV F/ 5 μg RSV F + 5 μg RSV F + dose/VRP-VCR2.1- 25 μg TLR7A/ 1 μg vA317u/ 1 μg vA317u/ vaccine RSV-F-full 5μg RSV F alum CNE17 RV01(36) F- 8215 1584 329326 159083 51705 specific4591 2910 440698 149037 76049 serum 3224 17153 421048 250812 80830 IgG17728 16701 272041 77892 90314 titers 3722 12119 368395 163630 120507 GMT5114 6932 360919 149942 80871

TABLE I-6 Continued from Table I-5. F-specific serum IgG1 titers ofBALB/c mice, 5 animals per group, intramuscular vaccinations on days 0and 21. Serum was collected for antibody analysis on day 35 (2wp2). Dataare represented as titers for individual mice and the geometric meantiters of 5 individual mice per group. dose/vaccine 5 μg 5 μg 5 μg 5 μg5 μg 5 μg 5 μg 5 μg 5 μg RSV F + RSV F + RSV F + RSV F + RSV F + RSV F +RSV F + RSV F + RSV F + RV01(36) RV01(36) RV01(36) 0.01 μg 0.1 μg 1 μg0.01 μg 0.1 μg 1 μg empty empty empty vA17u/ vA17u/ vA17u/ vA336u/vA336u/ vA336u/ (0.01 μg) (0.1 μg) (1 μg) RV01(36) RV01(36) RV01(36)RV01(36) RV01(36) RV01(36) F- 2974 5149 219778 11439 56323 135573 693776056 220841 specific 660 65459 513476 13986 55678 175715 55970 77182229310 serum 1892 8250 208658 22003 91464 187477 2327 101123 276811 IgG192 36886 242451 33160 100181 124329 8767 134221 268860 titers 3513 34019318148 51696 61527 290264 3786 197059 213310 GMT 1038 20349 283034 2270670712 174365 7859 109442 240461

TABLE I-7 F-specific serum IgG2a titers of BALB/c mice, 5 animals pergroup, intramuscular vaccinations on days 0 and 21. Serum was collectedfor antibody analysis on day 35 (2wp2). Data are represented as titersfor individual mice and the geometric mean titers of 5 individual miceper group. If an individual animal had a titer of <25 (limit ofdetection), it was assigned a titer of 5. 1E6 IU 5 μg RSV F/ 5 μg RSVF + 5 μg RSV F + dose/ VRP-VCR2.1- 25 μg TLR7A/ 1 μg vA317u/ 1 μgvA317u/ vaccine RSV-F-full 5 μg RSV F alum CNE17 RV01(36) F- 71930 579789 1489 299210 specific 66120 5 40013 6038 390179 serum 118752 539236750 5253 316071 IgG2a 117259 457 60506 13828 354418 titers 34487 551464 14135 396302 GMT 74428 31 74877 6209 349070

TABLE I-8 Continued from Table I-7. F-specific serum IgG2a titers ofBALB/c mice, 5 animals per group, intramuscular vaccinations on days 0and 21. Serum was collected for antibody analysis on day 35 (2wp2). Dataare represented as titers for individual mice and the geometric meantiters of 5 individual mice per group. If an individual animal had atiter of <25 (limit of detection), it was assigned a titer of 5.dose/vaccine 5 μg 5 μg 5 μg 5 μg 5 μg 5 μg 5 μg 5 μg 5 μg RSV F + RSVF + RSV F + RSV F + RSV F + RSV F + RSV F + RSV F + RSV F + RV01(36)RV01(36) RV01(36) 0.01 μg 0.1 μg 1 μg 0.01 μg 0.1 μg 1 μg empty emptyempty vA17u/ vA17u/ vA17u/ vA336u/ vA336u/ vA336u/ (0.01 μg) (0.1 μg) (1μg) RV01(36) RV01(36) RV01(36) RV01(36) RV01(36) RV01(36) F- 5 41 5 52540477 123780 282 73117 130968 specific 5 5 2397 10309 43203 58551 261873961 46273 serum 5 5 5 2278 68325 81341 284 47198 196773 IgG2a 5 268 57386 120738 115043 374 120915 138476 titers 630 34 5 18616 82748 15947471 93851 138895 GMT 13 25 17 4424 65370 101580 355 78050 118060

TABLE I-9 ratio of F-specific serum IgG2a:IgG1 titers FormulationsIgG2a:IgG1 ratio F protein (5 mcg), benchmark 1  1:220 Fprotein/Alum/TLR7A (25 mcg), benchmark 2 1:4.8 VRP (1E6), benchmark 310:1   F protein (5 mcg) + 1 mcg F RNA/Liposome 5:1  F protein (5 mcg) +1 mcg F RNA/CNE17 1:25  F protein (5 mcg) + 1 mcg GFP RNA/Liposome 1:1.7F protein (5 mcg) + 1 mcg GFP RNA/CNE17 1:2.0 F protein (5 mcg) + 0.01mcg equivalent of liposome 1:79  (without RNA) F protein (5 mcg) + 0.1mcg equivalent of liposome  1:388 (without RNA) F protein (5 mcg) + 1.0mcg equivalent of liposome  1:1204 (without RNA)

TABLE I-10 RSV serum neutralization titers of BALB/c mice, 5 animals pergroup, after intramuscular vaccinations on days 0 and 21. Serum wascollected for analysis on days 35 (2wp2) and 49 (4wp2). Data arerepresented as 60% plaque reduction neutralization titers of pools of 5mice, 1 pool per group. RSV serum neutralization titers dose/vaccine2wp2 4wp2 1E6 IU VRP-VCR2.1-RSV-F-full 53 62 5 μg RSV F <20 20 5 μg RSVF/25 μg TLR7A/alum 1002 1703 5 μg RSV F + 1 μg vA317u/CNE17 531 352 5 μgRSV F + 1 μg vA317u/RV01(36) 333 291 5 μg RSV F + RV01(36) empty (0.01μg) <20 24 5 μg RSV F + RV01(36) empty (0.1 μg) <20 30 5 μg RSV F +RV01(36) empty (1 μg) 74 108 5 μg RSV F + 0.01 μg vA17u/RV01(36) 27 34 5μg RSV F + 0.1 μg vA17u/RV01(36) 61 70 5 μg RSV F + 1 μg vA17u/RV01(36)133 113 5 μg RSV F + 0.01 μg vA336u/RV01(36) <20 <20 5 μg RSV F + 0.1 μgvA336u/RV01(36) 34 67 5 μg RSV F + 1 μg vA336u/RV01(36) 373 258 None <2023

TABLE I-11 Frequencies of RSV F-specific CD4+ splenic T cells on day 49(4wp2). Shown are net (antigen-specific) cytokine-positive frequency (%)± 95% confidence half-interval. Net frequencies shown in bold indicatestimulated responses that were statistically significantly >0. CD4+CD8−splenic T cells: % cytokine-positive and specific for RSV peptidesF51-66, F164-178, F309-323 dose/vaccine IFNg+ IL2+ IL5+ TNFa+ 1E6 IUVRP-VCR2.1-RSV-F- 0.10 ± 0.03 0.12 ± 0.05 −0.01 ± 0.02  0.13 ± 0.05 full5 μg RSV F 0.00 ± 0.01 0.03 ± 0.02 0.01 ± 0.01 0.00 ± 0.02 5 μg RSV F/25μg TLR7A/ 0.00 ± 0.01 0.04 ± 0.02 0.00 ± 0.01 0.02 ± 0.02 Alum 5 μg RSVF + 0.03 ± 0.01 0.10 ± 0.03 0.01 ± 0.01 0.06 ± 0.03 1 μg vA317u/CNE17 5μg RSV F + 0.12 ± 0.03 0.31 ± 0.05 0.01 ± 0.01 0.31 ± 0.05 1 μgvA317u/RV01(36) 5 μg RSV F + 0.00 ± 0.01 0.02 ± 0.01 0.00 ± 0.01 0.02 ±0.02 RV01(36) empty (0.01 μg) 5 μg RSV F + 0.00 ± 0.01 0.04 ± 0.02 0.02± 0.01 0.01 ± 0.02 RV01(36) empty (0.1 μg) 5 μg RSV F + 0.00 ± 0.01 0.06± 0.02 0.07 ± 0.02 0.01 ± 0.02 RV01(36) empty (1 μg) 5 μg RSV F + 0.01 ±0.01 0.06 ± 0.02 0.01 ± 0.01 0.01 ± 0.02 0.01 μg vA17u/RV01(36) 5 μg RSVF + 0.02 ± 0.01 0.09 ± 0.02 0.01 ± 0.01 0.06 ± 0.02 0.1 μgvA17u/RV01(36) 5 μg RSV F + 0.07 ± 0.02 0.21 ± 0.04 0.02 ± 0.02 0.18 ±0.04 1 μg vA17u/RV01(36) 5 μg RSV F + 0.01 ± 0.01 0.02 ± 0.01 0.00 ±0.01 0.01 ± 0.02 0.01 μg vA336u/RV01(36) 5 μg RSV F + 0.01 ± 0.01 0.06 ±0.02 0.00 ± 0.01 0.04 ± 0.02 0.1 μg vA336u/RV01(36) 5 μg RSV F + 0.10 ±0.03 0.22 ± 0.04 0.00 ± 0.01 0.22 ± 0.04 1 μg vA336u/RV01(36) None 0.00± 0.01 0.00 ± 0.01 0.00 ± 0.01 −0.01 ± 0.02 

TABLE I-12 Frequencies of RSV F-specific CD8+ splenic T cells on day 49(4wp2). Shown are net (antigen-specific) cytokine-positive frequency (%)± 95% confidence half-interval. Net frequencies shown in bold indicatestimulated responses that were statistically significantly >0. CD8+CD4−splenic T cells: % cytokine-positive and specific for RSV F peptidesF85-93 and F249-258 dose/vaccine IFNg+ IL2+ IL5+ TNFa+ 1E6 IUVRP-VCR2.1-RSV-F- 1.51 ± 0.16 0.59 ± 0.10 0.00 ± 0.02 1.11 ± 0.14 full 5μg RSV F −0.01 ± 0.06  0.03 ± 0.03 0.00 ± 0.01 0.00 ± 0.03 5 μg RSV F/25μg TLR7A/ 0.21 ± 0.07 0.16 ± 0.06 0.00 ± 0.02 0.22 ± 0.07 Alum 5 μg RSVF + 1.40 ± 0.15 0.74 ± 0.11 0.00 ± 0.03 1.03 ± 0.13 1 μg vA317u/CNE17 5μg RSV F + 5.60 ± 0.29 2.26 ± 0.18 0.00 ± 0.02 4.56 ± 0.26 1 μgvA317u/RV01(36) 5 μg RSV F + 0.11 ± 0.06 0.07 ± 0.04 −0.04 ± 0.03  0.07± 0.05 RV01(36) empty (0.01 μg) 5 μg RSV F + 0.19 ± 0.07 0.20 ± 0.060.00 ± 0.02 0.20 ± 0.06 RV01(36) empty (0.1 μg) 5 μg RSV F + 0.60 ± 0.100.66 ± 0.10 0.01 ± 0.02 0.66 ± 0.11 RV01(36) empty (1 μg) 5 μg RSV F +0.10 ± 0.05 0.06 ± 0.04 0.02 ± 0.03 0.07 ± 0.04 0.01 μg vA17u/RV01(36) 5μg RSV F + 1.00 ± 0.13 0.51 ± 0.09 −0.03 ± 0.02  0.72 ± 0.11 0.1 μgvA17u/RV01(36) 5 μg RSV F + 2.18 ± 0.18 1.24 ± 0.14 0.00 ± 0.02 1.67 ±0.16 1 μg vA17u/RV01(36) 5 μg RSV F + 0.12 ± 0.06 0.12 ± 0.04 0.01 ±0.02 0.13 ± 0.05 0.01 μg vA336u/RV01(36) 5 μg RSV F + 1.01 ± 0.13 0.54 ±0.10 0.01 ± 0.03 0.80 ± 0.12 0.1 μg vA336u/RV01(36) 5 μg RSV F + 2.39 ±0.19 1.16 ± 0.14 0.02 ± 0.03 1.93 ± 0.17 1 μg vA336u/RV01(36) None 0.00± 0.03 0.00 ± 0.02 −0.02 ± 0.03  0.02 ± 0.03

Example II—Co-Administration of RNA and Protein and SequentialAdministration of RNA and Protein to Mice

The vA142 replicon was used for this study. This construct expresses thefull-length wild type surface fusion glycoprotein of RSV with the fusionpeptide deleted and the 3′ end of the replicon is formed byribozyme-mediated cleavage. BALB/c mice, 116 total, were divided into 11groups (4-22 animals per group).

Group 1 (8 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with unadjuvanted RSV-F subunit proteinvaccine (3 μg) and all animals were sacrificed at day 42.

Group 2 (22 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with the RSV-F subunit protein vaccine (3 μg)adjuvanted with alum. 4 animals were sacrificed at day 42. 6 mice weregiven a third vaccination at day 42 with the RSV-F subunit proteinvaccine (3 μg) adjuvanted with alum and the animals were sacrificed atday 63. 6 mice were given a third vaccination at day 42 with RNA (vA142,1 μg) formulated with CNE17 (10:1 N:P ratio) and the animals weresacrificed at day 63. 6 mice were given a third vaccination at day 42with RNA (vA142, 1 μg) formulated in LNP (RV01(39) (composition: DlinDMA40%, DSPC—10%, Chol—48%, PEG DMG 5000-2% at an N:P ratio of 8:1, madeusing Method A, 175 μg RNA batch size) and the animals were sacrificedat day 63.

Group 3 (8 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with the RSV-F subunit protein vaccine (3 μg)adjuvanted with alum and a small molecule TLR7 agonist (TLR7A), allanimals were sacrificed at day 42.

Group 4 (8 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with the RSV-F subunit protein vaccine (3 μg)adjuvanted with MF59 (equivalent squalene dose to CNE17) and all animalswere sacrificed at day 42.

Group 5 (8 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with the RSV-F subunit protein vaccine (3 μg)adjuvanted with CNE17 and animals were sacrificed at day 42.

Group 6 (16 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with RNA (vA142, 1 μg) formulated with CNE17.4 animals were sacrificed at day 42. 6 mice were given a thirdvaccination at day 42 with the RSV-F subunit protein vaccine (3 μg)adjuvanted with alum and the animals were sacrificed at day 63. 6 micewere given a third vaccination at day 42 with RNA (vA142, 1 μg)formulated with CNE17 and the animals were sacrificed at day 63.

Group 7 (16 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with RNA (vA142, 1 μg) formulated with LNP(RV01(39)) (composition: DlinDMA 40%, DSPC—10%, Chol—48%, PEG DMG5000-2% at an N:P ratio of 8:1, made using Method A, 175 μg RNA batchsize). 4 animals were sacrificed at day 42. 6 mice were given a thirdvaccination at day 42 with the RSV-F subunit protein vaccine (3 μg)adjuvanted with alum and the animals were sacrificed at day 63. 6 micewere given a third vaccination at day 42 with RNA (vA142, 1 μg)formulated with LNP (RV01(39)) (composition: DlinDMA 40%, DSPC—10%,Chol—48%, PEG DMG 5000-2% at an N:P ratio of 8:1, made using Method A,175 μg RNA batch size) and the animals were sacrificed at day 63.

Group 8 (8 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with RNA (vA142, 1 μg) and the RSV-F proteinsubunit (no alum or MF59) formulated with CNE17. All animals weresacrificed at day 42.

Group 9 (8 animals) were given bilateral intramuscular vaccinations (50μL per leg) on days 0, 21 with RNA (vA142, 1 μg) formulated in LNP(RV01(39)) (composition: DlinDMA 40%, DSPC—10%, Chol—48%, PEG DMG5000-2% at an N:P ratio of 8:1, made using Method A, 175 μg RNA batchsize) and mixed with the RSV-F protein subunit (no alum or MF59). Allanimals were sacrificed at day 42.

Group 10 (10 animals) were given bilateral intramuscular vaccinations(50 μL per leg) on days 0, 21 with VRPs (1×10⁶ IU) expressing thefull-length wild type surface fusion glycoprotein of RSV with the fusionpeptide deleted. 4 animals were sacrificed at day 42. 6 mice were givena third vaccination at day 42 with the RSV-F subunit protein vaccine (3μg) adjuvanted with alum and the animals were sacrificed at day 63.

Group 11 (4 Animals) Naïve control.

Serum was collected for antibody analysis on days 0, 20, 41 and 63.Spleens were harvested on days 42 and 63 for T-cell analysis.

Results

F-specific serum IgG titers are shown in table II-1 (day 20), II-2 (day41) and II-7 (day 63). F-specific serum IgG1 titers are shown in tablesII-3 (day 41) and II-8 (day 63), and F-specific serum IgG2a titers areshown in tables II-4 (day 41) and II-9 (day 63). RSV serum neutralizingantibody titers are shown in tables II-5 (day 41) and II-10 (day 63).Average net F-specific cytokine-positive T cell frequencies (CD4+ orCD8+) are shown in tables II-6 (day 42) and II-11 (day 63).

Conclusions

The data collected after the second vaccination of BALB/c mice show thefollowing:

CNE17 is a potent adjuvant for the F subunit, as the F-specific IgGtiters induced by vaccination with F subunit+CNE17 were greater thanthose induced by unadjuvanted F subunit, and equal to those induced by Fsubunit+alum or F subunit+MF59. In each of these four groups (Fsubunit+CNE17, unadjuvanted F subunit, F subunit+alum, F subunit+MF59)the F-specific IgG response was primarily IgG1 (very little/no IgG2a).Addition of a TLR7 agonist (TLR7A) to the F+alum vaccine increased themagnitude of the total F-specific IgG response by approximatelytwo-fold, and induced class switching to IgG2a.

vA142+CNE17 and vA142+RV01 vaccines each induced F-specific IgG classswitching to IgG2a (F-specific IgG2a titers were at least ten-foldgreater than F-specific IgG1 titers when measured after the secondvaccination), although the magnitude of the response to vA142+RV01 wasapproximately ten-fold greater response than the response tovA142+CNE17.

vA142+CNE17+F subunit (without alum or MF59) induced F-specific IgGtiters that were 24-fold those induced by vA142+CNE17 (without Fsubunit), but were approximately equal to those induced by Fsubunit+CNE17 (without vA142). The F-specific IgG isotype induced byvA142+CNE17+F subunit was primarily IgG1, matching the response to Fsubunit+CNE17 (primarily IgG1) but not the response to vA142+CNE17(primarily IgG2a).

vA142+RV01+F subunit (without alum or MF59) induced a F-specific IgGtiter that was approximately equal in magnitude to that induced byvA142+CNE17 (without F subunit). The F-specific IgG isotype induced bythe vA142+RV01+F subunit vaccine was primarily IgG2a, matching theresponse to vA142+CNE17.

The RSV neutralization titers at the two weeks post second vaccinationtime point correlated well with the total F-specific IgG titers.

Splenic T cell responses (CD4 and CD8) to all subunit vaccines that didnot contain replicon were low. Replicon vaccines induced a robust CD8response, and a moderate CD4 response. Both T cell subsets producedprimarily type 1 cytokines including IFNγ, TNFα, and IL2. The magnitudeof the CD8 T cell response to vA142+RV01 was approximately equal to theresponse induced by VRP, and about five-fold greater than the responseinduced by vA142+CNE17.

vA142+CNE17+F subunit induced a robust CD8 T cell response that wasgreater in magnitude than the sum of the responses induced by Fsubunit+CNE17 and vA142+CNE17 vaccines. vA142+RV01+F subunit induced arobust CD8 response that was equal in magnitude to that induced byvA142+RV01.

The data collected after the third vaccination of BALB/c mice show thefollowing:

A third vA142+CNE17 vaccination boosted the F-specific IgG and RSVneutralization titers three fold. A third vA142+RV01 vaccination boostedthe F-specific IgG titer two fold and the RSV neutralization titer fourfold.

F-specific and RSV neutralization titers were greatly enhanced after Fsubunit+alum vaccination of mice previously vaccinated twice withvA142+CNE17 (increased IgG titers by 46 fold and RSV neutralizationtiters by 62 fold). F-specific and RSV neutralization titers were alsoenhanced after F subunit+alum vaccination of mice previously vaccinatedtwice with vA142+RV01 (increased IgG titers by 5 fold and RSVneutralization titers by 6 fold). The magnitude of the RSVneutralization titers after two replicon vaccinations followed by one Fsubunit+alum vaccination were equal to or greater than that induced bythree subunit vaccinations.

After two vaccinations, the F-specific IgG isotype induced by Fsubunit+alum was primarily IgG1, whereas the isotype induced byvA142+CNE17 or vA142+RV01 was primarily IgG2a. This isotype balance setby the priming vaccinations was maintained after a third vaccinationwith a homologous or heterologous vaccine. For example, the dominantF-specific IgG isotype was IgG1 for mice vaccinated three times with Fsubunit+alum, vaccinated twice with F subunit+alum and then once withvA142+CNE17, or vaccinated twice with F subunit+alum and then once withvA142+RV01. The dominant F-specific IgG isotype was IgG2a for micevaccinated three times with vA142+CNE17, mice vaccinated three timeswith vA142+RV01, mice vaccinated twice with vA142+CNE17 and then oncewith F subunit+alum, and mice vaccinated twice with vA142+RV01 and thenonce with F subunit+alum. IgG1 is often associated with a T helper type1 (Th1) and IgG2a is often associated with a T helper type 2 (Th2)response. These data show that replicon vaccines induced a Th1-typeresponse that was maintained even after a subunit+alum boost (that initself induces a Th2-like response).

TABLE II-1 F-specific serum IgG titers of BALB/c mice, 2-22 animals pergroup, 20 days (~3wp1) after intramuscular vaccination with theindicated vaccines. Data are represented as titers for individualanimals and the geometric mean titer (bottom row in table) of eachgroup. If an individual animal had a titer of <25 (limit of detection)it was assigned a titer of 5. ~3wp1 (day 20) F-specific serum IgG titersGroup 3 ug F 3 ug F 3 ug F 3 ug F trimer trimer trimer trimer 3 ug Fsubunit + subunit + 3 ug F subunit + subunit + 3 ug F trimer alum + ⅓trimer 1 ug 1 ug 1 ug 1 ug 1E6 IU trimer subunit + 25 ug dose subunit +vA142 + vA142 + vA142 + vA142 + VRP subunit alum TLR7A MF59 CNE17 CNE17RV01 (39) CNE17 RV01 (39) (A142) naive 5 2792 10085 2169 2753 1019 50782561 3622 5591 5 128 3337 14233 2046 1911 388 3995 1309 6320 3304 5 824533 19072 2817 3158 1167 11053 1639 5060 4378 1414 3182 6711 5209 19131977 6199 2778 6295 6363 46 4991 21158 1509 2247 731 6295 2058 5158 2349370 2902 12073 1445 2404 897 4503 4365 5027 5251 114 3480 10915 19361615 500 8196 5393 6767 5419 384 3370 19626 28213 2130 885 5225 58947459 2965 4444 653 7301 3856 2567 854 4438 4493 3005 1335 5142 4352 95151197 3949 2875 967 5833 2775 1908 8050 3918 1676 2179 1654 388 3298 50785410 5950 5159 7033 3552 124 3862 13344 3063 2220 924 5289 2850 55914227 5

TABLE II-2 F-specific serum IgG titers of BALB/c mice, 2-22 animals pergroup, after intramuscular vaccination with the indicated vaccines ondays 0 and 21. Serum was collected for antibody analysis on day 41(~3wp2). Data are represented as titers for individual animals and thegeometric mean titer (bottom row of table) for each group. If anindividual animal had a titer of <25 (limit of detection) it wasassigned a titer of 5. ~3wp2 (day 41) F-specific serum IgG titers Group3 ug F 3 ug F 3 ug F 3 ug F trimer trimer trimer trimer 3 ug F subunit +subunit + 3 ug F subunit + subunit + 3 ug F trimer alum + ⅓ trimer 1 ug1 ug 1 ug 1 ug 1E6 IU trimer subunit + 25 ug dose subunit + vA142 +vA142 + vA142 + vA142 + VRP subunit alum TLR7A MF59 CNE17 CNE17 RV01(39) CNE17 RV01 (39) (A142) naive 3333 127489 371967 143371 220170 631141898 191234 48414 45898 5 64917 126823 279675 113913 119981 13605 72032155411 107539 55587 5 18031 272342 442396 125447 111939 3550 114060140117 89808 80201 44805 163175 372514 226022 176715 9278 98383 19516593729 24167 27313 205032 419241 166683 79338 7292 63614 181607 8175426529 67566 153234 304957 140631 108340 13343 78069 193053 62075 2452911077 89144 412571 213628 172781 2963 64790 250282 82313 32024 66825208898 212292 363933 232211 15017 65534 212180 126066 35789 163304 1223672860 17290 328763 18115 93245 31304 186277 16334 78355 254714 544635923 199791 1288 129992 70668 13656 133508 194242 11395 30533 18707018314 67121 268138 1962 137072 205685 85988 333357 352752 26439 180136343088 174095 143540 7879 71680 187277 83253 33886 5

TABLE II-3 F-specific serum IgG1 titers of BALB/c mice, 2-22 animals pergroup, after intramuscular vaccination with the indicated vaccines ondays 0 and 21. Serum was collected for antibody analysis on day 41(~3wp2). Data are represented as titers for individual animals and thegeometric mean titer (bottom row of table) for each group. If anindividual animal had a titer of <25 (limit of detection) it wasassigned a titer of 5. ~3wp2 (day 41) F-specific serum IgG1 titers Group3 ug F 3 ug F 3 ug F 3 ug F trimer trimer trimer trimer 3 ug F subunit +subunit + 3 ug F subunit + subunit + 3 ug F trimer alum + ⅓ trimer 1 ug1 ug 1 ug 1 ug 1E6 IU trimer subunit + 25 ug dose subunit + vA142 +vA142 + vA142 + vA142 + VRP subunit alum TLR7A MF59 CNE17 CNE17 RV01(39)CNE17 RV01 (39) (A142) naive 4751 225245 350032 266739 903304 1650 14319451989 17066 13904 5 107732 238261 271960 178609 197261 4957 9623 41836022116 9898 5 28126 650444 367427 219141 207527 1238 30034 279866 655285531 59285 350077 315508 581887 241267 936 14032 449010 21682 5097 36991522278 360452 343140 229090 5016 8154 347235 32735 10355 86261 285165288697 224815 191527 519 6857 408570 17214 3932 15630 132721 648766531617 321951 7008 23195 446552 19802 4090 86314 366415 106856 506364457173 3454 8078 561199 39707 5493 294116 4947 27901 972 940674 396025553 2990 333129 2330 17643 302960 552 6776 352372 504 57247 94693 186410066 304688 5340 7894 293133 5 10994 298757 180582 317663 109094 388756609926 37208 300542 307342 325547 295168 1449 14154 412900 26423 5027 5

TABLE II-4 F-specific serum IgG2a titers of BALB/c mice, 2-22 animalsper group, after intramuscular vaccination with the indicated vaccineson days 0 and 21. Serum was collected for antibody analysis on day 41(~3wp2). Data are represented as titers for individual animals and thegeometric mean titer (bottom row of table) for each group. If anindividual animal had a titer of <125 (limit of detection) it wasassigned a titer of 25. ~3wp2 (day 41) F-specific serum IgG2a titersGroup 3 ug F 3 ug F 3 ug F 3 ug F trimer trimer trimer trimer 3 ug Fsubunit + subunit + 3 ug F subunit + subunit + 3 ug F trimer alum + ⅓trimer 1 ug 1 ug 1 ug 1 ug 1E6 IU trimer subunit + 25 ug dose subunit +vA142 + vA142 + vA142 + vA142 + VRP subunit alum TLR7A MF59 CNE17 CNE17RV01 (39) CNE17 RV01 (39) (A142) naive 25 25 96017 248 25 20432 8709336243 109284 119958 25 25 25 18204 5412 25 20469 194006 2525 298231153137 25 25 25 131509 1467 25 5716 414280 1440 113622 174041 1484 2569704 25 25 42031 340224 529 179564 64698 479 25 138075 25 1660 26914172298 17489 148624 64977 2326 25 79634 25 25 11979 240916 25 13529564744 662 2631 71358 25 37663 27314 218653 25 179619 140264 8085 25195149 319060 25 28250 256190 5480 268365 101520 25 35361 163792 6147825 33064 248366 89519 25 16448 179106 25 4934 127192 25 49826 356910 31827778 598570 410 34555 59459 25 6434 150814 25 25 25 25 25 1268 329 4783891 354 105 20135 205134 1195 168328 96244 25

TABLE II-5 RSV serum neutralization titers of BALB/c mice afterintramuscular vaccinations with the indicated vaccines on days 0 and 21.Serum was collected for analysis on day 41 (~3wp2). Data are representedas 60% RSV neutralization titers for pools of 3-4 animals per group andgeometric mean titers of 1-7 pools per group. If an individual pool hada titer of <20 (limit of detection) it was assigned a titer of 10. RSVserum neutralization titers ~3wp2 (day 41) Group pool 1 pool 2 pool 3pool 4 pool 5 pool 6 pool 7 GMT 3 ug F trimer subunit 10 10 3 ug Ftrimer subunit + alum 159 221 655 562 396 198 303 315 3 ug F trimersubunit + alum + 25 ug TLR7A 595 214 357 3 ug F trimer subunit + ⅓ doseMF59 509 71 190 3 ug F trimer subunit + CNE17 106 149 126 1 ug vA142 +CNE17 29 38 34 31 41 34 1 ug vA142 + RV01 (39) 344 1995 311 148 331 4023 ug F trimer subunit + 1 ug vA142 + CNE17 73 352 160 3 ug F trimersubunit + 1 ug vA142 + RV01 (39) 116 262 174 1E6 IU VRP (A142) 82 61 4058

TABLE II-6 Frequencies of RSV F-specific CD4+ or CD8+ splenic T cells ofBALB/c mice, 4 animals per group (except 2 in naive group), afterintramuscular vaccination with the indicated vaccines on days 0 and 21.Spleens were collected for T cell analysis on day 42 (~3wp2). Shown areaverage net F-specific cytokine- positive frequencies (%) of duplicatewells of one pool of 4 spleens per group. ~3wp2 (day 41) splenicF-specific T cell response CD4+CD8− CD4−CD8+ Group IFNg+ TNFa+ IL-2+IL-5+ IFNg+ TNFa+ IL-2+ IL-5+ 3 ug F trimer subunit 0.00 0.01 0.00 0.010.01 0.01 0.01 0.00 3 ug F trimer subunit + 0.00 0.00 0.02 0.00 0.010.00 0.04 0.00 alum 3 ug F trimer subunit + 0.01 0.02 0.03 0.01 0.020.04 0.03 0.01 alum + 25 ug TLR7A 3 ug F trimer subunit + 0.02 0.02 0.050.02 0.09 0.10 0.24 0.00 ⅓ dose MF59 3 ug F trimer subunit + 0.01 0.020.04 0.05 0.02 0.10 0.14 0.00 CNE17 1 ug vA142 + CNE17 0.05 0.08 0.160.01 0.57 0.59 0.37 0.00 1 ug vA142 + RV01 (39) 0.09 0.18 0.34 0.00 3.453.59 1.41 0.01 3 ug F trimer subunit + 0.04 0.08 0.19 0.09 2.42 2.550.99 0.01 1 ug vA142 + CNE17 3 ug F trimer subunit + 0.13 0.26 0.50 0.003.35 3.32 1.92 0.01 1 ug vA142 + RV01 (39) 1E6 IU VRP (A142) 0.10 0.180.30 0.01 2.68 2.93 1.55 0.00 Naïve 0.00 0.01 0.01 0.00 0.00 0.00 0.000.00

TABLE II-7 F-specific serum IgG titers of BALB/c mice, 2-6 animals pergroup, after intramuscular vaccinations with the indicated 1^(st),2^(nd), and 3^(rd) vaccines (administered on days 0, 21, and 42,respectively). Serum was collected for antibody analysis on day 63(3wp3). Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <25 (limit of detection) it was assigned a titer of 5. Group 1^(st)and 2^(nd) vaccination 3^(rd) vaccination 3wp3 (day 63) F-specific serumIgG titers GMT 3 ug F trimer subunit + 3 ug F trimer subunit + 397125219348 234682 474062 325653 485166 339498 alum alum 3 ug F trimersubunit + 1 ug vA142 + CNE17 295650 285307 301188 108903 191240 263752227749 alum 3 ug F trimer subunit + 1 ug vA142 + RV01 (39) 389432 220866340951 263722 316715 579546 335242 alum 1 ug vA142 + CNE17 3 ug F trimersubunit + 378695 368173 638358 580096 535790 747680 523925 alum 1 ugvA142 + CNE17 1 ug vA142 + CNE17 28392 25370 20404 25687 28161 551519710 1 ug vA142 + RV01 3 ug F trimer subunit + 422179 446249 284067287956 441567 506771 388691 alum 1 ug vA142 + RV01 (39) 1 ug vA142 +RV01 (39) 124230 68942 135140 161575 81778 99206 107195 1E6 IU VRP(A142) 3 ug F trimer subunit + 560903 458740 254037 268794 491496 200132346419 alum naive Naïve 5 5 5

TABLE II-8 F-specific serum IgG1 titers of BALB/c mice, 2-6 animals pergroup, after intramuscular vaccinations with the indicated 1^(st),2^(nd), and 3^(rd) vaccines (administered on days 0, 21, and 42,respectively). Serum was collected for antibody analysis on day 63(3wp3). Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <125 (limit of detection) it was assigned a titer of 25. Group 1^(st)and 2^(nd) vaccination 3^(rd) vaccination 3wp3 (day 63) F-specific serumIgG1 titers GMT 3 ug F trimer subunit + 3 ug F trimer subunit + 472727245635 204730 475968 378693 569982 366974 alum alum 3 ug F trimersubunit + 1 ug vA142 + CNE17 332385 331830 322523 118291 189876 261341243571 alum 3 ug F trimer subunit + 1 ug vA142 + RV01 (39) 466588 291761417428 288690 350836 598125 388578 alum 1 ug vA142 + CNE17 3 ug F trimersubunit + 110372 97254 502079 172198 242153 79623 161727 alum 1 ugvA142 + CNE17 1 ug vA142 + CNE17 8395 1386 488 8881 12117 25 1575 1 ugvA142 + RV01 3 ug F trimer subunit + 54308 155078 153748 32589 193390105775 97578 alum 1 ug vA142 + RV01 (39) 1 ug vA142 + RV01 (39) 255657668 51279 8030 18916 25651 18428 1E6 IU VRP (A142) 3 ug F trimersubunit + 304345 235517 20501 53684 105506 18831 73427 alum naive Naïve25 25 25

TABLE II-9 F-specific serum IgG2a titers of BALB/c mice, 2-6 animals pergroup, after intramuscular vaccinations with the indicated 1^(st),2^(nd), and 3^(rd) vaccines (administered on days 0, 21, and 42,respectively). Serum was collected for antibody analysis on day 63(3wp3). Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <125 (limit of detection) it was assigned a titer of 25. Group 1^(st)and 2^(nd) vaccination 3^(rd) vaccination 3wp3 (day 63) F-specific serumIgG2a titers GMT 3 ug F trimer subunit + 3 ug F trimer subunit + 25 255124 25 683 495 173 alum alum 3 ug F trimer subunit + 1 ug vA142 + CNE1725 678 25 1789 2090 52 209 alum 3 ug F trimer subunit + 1 ug vA142 +RV01 (39) 7279 4583 8964 10947 3749 10965 7158 alum 1 ug vA142 + CNE17 3ug F trimer subunit + 544099 598814 601179 241979 529568 1529305 580802alum 1 ug vA142 + CNE17 1 ug vA142 + CNE17 29369 27933 38983 19630 2776314863 25248 1 ug vA142 + RV01 (39) 3 ug F trimer subunit + 886323 471796277106 837672 628141 852851 610941 alum 1 ug vA142 + RV01 (39) 1 ugvA142 + RV01 (39) 210624 150131 198056 340986 122466 140132 182253 1E6IU VRP (A142) 3 ug F trimer subunit + 621104 472535 674444 548436 824451581684 611064 alum naive Naïve 25 25 25

TABLE II-10 RSV serum neutralization titers of BALB/c mice afterintramuscular vaccinations with the indicated 1^(st), 2^(nd), and 3^(rd)vaccines (administered on days 0, 21, and 42, respectively). Serum wascollected for analysis on day 63 (3wp3). Data are represented as 60% RSVneutralization titers for 2 pools of 3 animals per group and geometricmean titers of these 2 pools per group. If an individual pool had atiter of <20 (limit of detection) it was assigned a titer of 10. GroupRSV serum neutralization titers 1^(st) and 2^(nd) 3wp3 (day 63)vaccination 3^(rd) vaccination pool 1 pool 2 GMT 3 ug F trimer 3 ug Ftrimer 539 2982 1268 subunit + alum subunit + alum 3 ug F trimer 1 ugvA142 + 1199 1507 1344 subunit + alum CNE17 3 ug F trimer 1 ug vA142 +1943 4024 2796 subunit + alum RV01 (39) 1 ug vA142 + 3 ug F trimer 22832169 2225 CNE17 subunit + alum 1 ug vA142 + 1 ug vA142 + 144 96 118CNE17 CNE17 1 ug vA142 + 3 ug F trimer 4654 4882 4767 RV01 (39)subunit + alum 1 ug vA142 + 1 ug vA142 + 530 1240 868 RV01 (39) RV01(39) 1E6 IU VRP 3 ug F trimer 4672 2287 3269 (A142) subunit + alum naiveNaïve 10 10

TABLE II-11 Frequencies of RSV F-specific CD4+ or CD8+ splenic T cellsof BALB/c mice, 6 animals per group (except 2 in naive group), afterintramuscular vaccinations with the indicated 1^(st), 2^(nd), and 3^(rd)vaccines (administered on days 0, 21, and 42, respectively). Spleenswere collected for T cell analysis on day 63 (3wp3). Shown are averagenet F-specific cytokine- positive frequencies (%) of duplicate wells ofone pool of 6 spleens per group. Group 3wp2 (day 63) splenic F-specificT cell response 1^(st) and 2^(nd) CD4+CD8− CD4−CD8+ vaccination 3^(rd)vaccination IFNg+ TNFa+ IL-2+ IL-5+ IFNg+ TNFa+ IL-2+ IL-5+ 3 ug Ftrimer 3 ug F trimer 0.00 0.01 0.02 0.02 0.01 0.01 0.03 0.00 subunit +alum subunit + alum 3 ug F trimer 1 ug vA142 + 0.00 0.03 0.07 0.02 0.140.23 0.26 0.01 subunit + alum CNE17 3 ug F trimer 1 ug vA142 + 0.00 0.040.07 0.01 0.09 0.19 0.18 0.00 subunit + alum RV01 (39) 1 ug vA142 + 3 ugF trimer 0.07 0.29 0.34 0.01 0.37 0.49 0.25 0.01 CNE17 subunit + alum 1ug vA142 + 1 ug vA142 + 0.02 0.16 0.19 0.00 0.29 0.35 0.28 0.01 CNE17CNE17 1 ug vA142 + 3 ug F trimer 0.03 0.18 0.19 0.01 0.97 1.47 0.51 0.00RV01 (39) subunit + alum 1 ug vA142 + 1 ug vA142 + 0.05 0.37 0.35 0.000.99 1.47 0.58 0.00 RV01 (39) RV01 (39) 1E6 IU VRP 3 ug F trimer 0.050.19 0.18 0.00 1.33 1.75 0.56 0.02 (A142) subunit + alum naive Naïve0.00 0.00 0.00 0.01 0.01 0.01 0.02 0.02

Example III—Sequential Administration of RNA and Protein to Rats (Study1)

Three different replicons were used for this study: the vA317 replicon,which expresses the full-length wild type surface fusion glycoprotein ofRSV (RSV-F); the vA318 replicon, which expresses the truncated(transmembrane and cytoplasmic tail removed) surface fusion glycoproteinof RSV; and the vA142 replicon, which expresses the full-length wildtype surface fusion glycoprotein of RSV with the fusion peptide deleted.Cotton rats, 2-8 animals per group, were given intramuscularvaccinations (100 μL in one leg) on days 0 and 21 with the threedifferent RNAs (vA317, vA318, vA142) formulated in LNPs (RV01(29) orCNE17 (N:P ratio 10:1) and given at two doses (1.0 and 0.1 μg, 8animals/group). LNPs had the following composition: 40% DlinDMA, 10%DSPC, 48% Chol, 2% PEG DMG 2000, N:P ratio of 8:1 and was made usingMethod B, except a 150 μg RNA batch size was used. Control groupsreceived the RSV-F subunit protein vaccine (5 μg) adjuvanted with alum(8 animals/group) and VRP's expressing full-length RSV-F (1×10⁶ IU, 8animals/group). All the LNP, subunit and VRP groups received a thirdvaccination (day 56) with RSV-F subunit protein vaccine (5 μg)adjuvanted with alum. In addition there was a naïve control (4animals/group). Serum was collected for antibody analysis on days 0, 21,35, 56, 70. In addition, two groups were given bilateral intramuscularvaccinations (50 μL per leg) on days 0 and 56 with RNA (vA317, 1 μg)formulated in LNPs (RV01(29) or CNE17 (8 animals/group). These groupsdid not receive a third vaccination with the subunit protein vaccine.Serum was collected for antibody analysis on days 0, 14, 21, 28, 35, 42,56, 70.

Results

F-specific serum IgG titers are shown in table III-1 (day 21), III-2(day 35), III-3 (day 56), III-4 (day 70) for the groups that receivedthe third vaccination with the RSV-F subunit vaccine. RSV serumneutralizing antibody titers on days 21, 35, 56 and 70 are shown intable III-5. F-specific serum IgG titers on days 14, 21, 28, 35, 42, 56,70 are shown in table III-6 for the two groups that did not receive thethird vaccination with the RSV-F subunit vaccine. RSV serum neutralizingantibody titers on days 14, 21, 28, 35, 42, 56, 70 for these two groupsare shown in table III-7.

Conclusions

When formulated with RV01 or CNE17, all three replicons evaluated inthis study (vA317, vA318, vA142) were immunogenic in cotton rats. Eachelicited serum F-specific IgG and RSV neutralizing antibodies after thefirst vaccination, and a second vaccination effectively boosted theresponse. F-specific IgG titers after the second vaccination with 1.0 μgreplicon were 1.5 to 4-fold higher than after the second vaccinationwith 0.1 μg replicon, regardless of the formulation. The three repliconsevaluated in this study (vA317, vA318, vA142) elicited comparableantibody titers suggesting that full length RSV-F (with and without thefusion peptide) and truncated RSV-F are equally immunogenic in cottonrats. There was also little difference in antibody titer when the samereplicon was formulated with RV01 or CNE17, suggesting that these twoformulations are equally potent in cotton rats. RSV serum neutralizationtiters after a second replicon vaccination were at least ten-fold lowerthan after a second RSV-F subunit+alum or VRP vaccination.

Cotton rats vaccinated with replicon, VRP, or subunit on days 0 and 21were all vaccinated with 5 μg RSV-F trimer subunit+alum on day 56 (fiveweeks after the second vaccination). This third vaccination did notboost antibody titers in cotton rats previously vaccinated with F trimersubunit+alum, but it provided a large boost to titers in cotton ratspreviously vaccinated with replicon (both RV01 and CNE17 formulations).In most cases the RSV serum neutralization titers after two repliconvaccinations followed by an F subunit+alum boost were equal to orgreater than titers induced by two or three sequential F subunit+alumvaccinations.

For each group and time point a ratio of F-specific IgG titer to RSVneutralizing titer can be calculated, and is one indication of thequality of the antibody response. A lower ratio suggests that a largerfraction of the vaccine-induced antibody is capable of neutralizing RSV,and is therefore functional. The data from this study show that theF-specific IgG:RSV neutralizing antibody titer ratio was lower aftervaccination with replicon or VRP than after vaccination with Fsubunit+alum. The lower F-specific IgG:RSV neutralizing antibody titerratio set by two replicon or VRP vaccinations was maintained after a Fsubunit+alum boost.

This study also evaluated the kinetics of the antibody response to 1.0μg vA317 formulated with RV01 or CNE17. F-specific serum IgG and RSVneutralization titers induced by a single vaccination reached their peakaround day 21 and were maintained through at least day 56 (50-70% dropin F-specific IgG titer, little change in RSV neutralization titer). Ahomologous second vaccination was given to these animals on day 56, andboosted antibody titers to a level at least equal to that achieved whenthe second vaccination was administered on day 21.

TABLE III-1 F-specific serum IgG titers of cotton rats, 2-8 animals pergroup, 21 days after intramuscular vaccination with the indicatedvaccines. Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <25 (limit of detection) it was assigned a titer of 5. Group 3wp1(day 21) F-specific serum IgG titers GMT 1 ug vA318/RV01 (29) 503 750144 141 104 509 103 501 260 0.1 ug vA318/RV01 (29) 47 109 141 40 120 180161 68 95 1 ug vA318/CNE17 446 1084 466 174 258 537 233 656 412 0.1 ugvA318/CNE17 696 906 327 242 418 173 438 63 316 1 ug vA142/RV01 (29) 592379 1000 327 500 270 672 447 483 0.1 ug vA142/RV01 (29) 177 316 673 404175 448 183 439 314 1 ug vA142/CNE17 423 439 201 567 229 625 992 315 4190.1 ug vA142/CNE17 402 263 586 230 268 190 199 555 308 1 ug vA317/RV01(29) 379 892 373 851 1062 1453 1512 1000 841 1E6 VRP (F-full) 2448 16272057 2767 1677 1879 3891 1238 2075 5 ug F trimer subunit/alum 8433 137205940 7794 27047 26852 10612 16237 12685 Naïve 5 5 5

TABLE III-2 F-specific serum IgG titers of cotton rats, 2-8 animals pergroup, after intramuscular vaccination with the indicated vaccines ondays 0 and 21. Serum was collected for antibody analysis on day 35(2wp2). Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <25 (limit of detection) it was assigned a titer of 5. Group 2wp2(day 35) F-specific serum IgG titers GMT 1 ug vA318/RV01 (29) 1500 24471358 586 322 1319 1152 864 1027 0.1 ug vA318/RV01 (29) 219 197 317 560330 340 237 154 274 1 ug vA318/CNE17 4366 2334 2281 1273 2280 1178 4222009 1693 0.1 ug vA318/CNE17 3268 1485 1161 826 772 919 2688 489 1201 1ug vA142/RV01 (29) 5079 1013 2300 1178 2317 1388 2036 1485 1847 0.1 ugvA142/RV01 (29) 664 748 1085 2670 403 874 763 857 871 1 ug vA142/CNE172395 2025 1889 6150 1585 4536 3259 2791 0.1 ug vA142/CNE17 1446 298 30211745 2135 543 1152 1867 1242 1 ug vA317/RV01 (29) 2121 3478 1939 28337048 9963 7064 3475 4032 1E6 VRP (F-full) 3685 3953 5080 4517 1769 31533080 10074 3938 5 ug F trimer subunit/alum 49635 98715 45626 55704 6343749799 36424 54526 Naïve 5 5 5

TABLE III-3 F-specific serum IgG titers of cotton rats, 2-8 animals pergroup, after intramuscular vaccination with the indicated vaccines ondays 0 and 21. Serum was collected for antibody analysis on day 56(5wp2). Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <25 (limit of detection) it was assigned a titer of 5. Group 5wp2(day 56) F-specific serum IgG titers GMT 1 ug vA318/RV01 (29) 672 559421 136 135 313 399 407 332 0.1 ug vA318/RV01 (29) 95 166 163 81 192 313120 122 144 1 ug vA318/CNE17 1239 1208 475 497 1398 754 247 1076 749 0.1ug vA318/CNE17 1608 809 533 475 365 414 998 136 535 1 ug vA142/RV01 (29)1361 652 2068 634 2811 772 1426 709 1124 0.1 ug vA142/RV01 (29) 213 435663 1063 158 670 437 311 418 1 ug vA142/CNE17 824 841 615 2296 859 15221464 1094 0.1 ug vA142/CNE17 618 183 784 492 617 374 495 799 501 1 ugvA317/RV01 (29) 729 1822 709 1824 1523 2638 3088 925 1452 1E6 VRP(F-full) 1377 1493 1711 1716 1052 1244 1170 4569 1596 5 ug F trimersubunit/alum 23322 42848 25468 18597 27586 25058 23551 25846 Naïve 5 5 5

TABLE III-4 F-specific serum IgG titers of cotton rats, 2-8 animals pergroup, after intramuscular vaccination on days 0 and 21 with theindicated vaccines. All cotton rats (except those in naive group) werealso vaccinated with 5 μg of F trimer subunit/alum on day 56. Serum wascollected for antibody analysis on day 70 (2wp3). Data are representedas titers for individual animals and the geometric mean titer for eachgroup. If an individual animal had a titer of <25 (limit of detection)it was assigned a titer of 5. Group 2wp3 (day 70) F-specific serum IgGtiters GMT 1 ug vA318/RV01 (29) 26193 19838 14953 6158 9324 12742 1958315384 14263 0.1 ug vA318/RV01 (29) 922 2275 2385 962 2216 1674 2365 64822017 1 ug vA318/CNE17 14101 35083 19054 14272 35651 26929 8220 3731421122 0.1 ug vA318/CNE17 36218 37967 22406 20415 12633 8447 35167 467718004 1 ug vA142/RV01 (29) 14004 8393 16986 16213 11520 5990 15827 684611168 0.1 ug vA142/RV01 (29) 5584 12724 14350 17334 6566 15322 128499538 11023 1 ug vA142/CNE17 11441 13405 17436 17696 18594 20947 2241017016 0.1 ug vA142/CNE17 18417 24557 12250 25534 25210 15650 18176 2107419554 1 ug vA317/RV01 (29) 7825 17564 11108 17105 10895 11840 2511313684 13852 1E6 VRP (F-full) 7872 11490 17166 14129 15972 14840 2817213892 14574 5 ug F trimer subunit/alum 36631 75674 51823 41784 5971237095 50029 48864 Naïve 5 5 5

TABLE III-5 RSV serum neutralization titers of cotton rats, 2-8 animalsper group (2 per group for naive) after intramuscular vaccinations ondays 0 and 21 with the indicated vaccines followed by 5 ug F trimersubunit/alum for all groups (except naive) on day 56. Serum wascollected for analysis on days 21 (3wp1), 35 (2wp2), 56 (5wp2), and 70(2wp3). Data are represented as 60% RSV neutralization titers for 2pools of 3-4 animals per group and geometric mean titers of these 2pools per group (one pool of 2 animals for naive group). If anindividual pool had a titer of <20 (limit of detection) it was assigneda titer of 10. RSV serum neutralization titers 3wp1 (D 21) 2wp2 (D 35)5wp2 (D 56) 2wp3 (D 70) group pool 1 pool 2 GMT pool 1 pool 2 GMT pool 1pool 2 GMT pool 1 pool 2 GMT 1 ug vA318/RV01 51 65 58 194 92 134 112 110111 8446 3381 6344 (29) 0.1 ug vA318/RV01 33 50 41 112 92 102 87 45 635514 8013 6647 (29) 1 ug vA318/CNE17 60 68 64 278 176 221 139 137 1389640 6157 7704 0.1 ug vA318/CNE17 90 65 76 188 374 265 62 153 97 62144274 5154 1 ug vA142/RV01 69 86 77 243 476 340 113 360 202 3510 83905427 (29) 0.1 ug vA142/RV01 33 38 35 68 63 65 59 53 56 3123 1582 2223(29) 1 ug vA142/CNE17 41 139 75 315 374 343 246 128 177 7802 8083 79410.1 ug vA142/CNE17 30 68 45 122 142 132 59 217 113 5011 3424 4142 1 ugvA317/RV01 38 10 19 220 381 290 166 240 200 3627 4837 4189 (29) 1E6 VRP(F-full) 77 141 104 996 2379 1539 521 598 558 1329 6222 2876 5 ug Ftrimer 294 683 448 4029 4931 4457 1613 1647 1630 4604 2863 3631subunit/alum naive 10 10 10 10 10 10

TABLE III-6 F-specific serum IgG titers of cotton rats, 5 animals pergroup, after intramuscular vaccination on days 0 and 56 with theindicated vaccines. Serum was collected for antibody analysis on days 14(2wp1), 21 (3wp1), 28 (4wp1), 35 (5wp1), 42 (6wp1), 56 (8wp1) and 70(2wp2). Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <25 (limit of detection) it was assigned a titer of 5. F-specificserum IgG titers 2wp1 3wp1 4wp1 5wp1 6wp1 8wp1 2wp2 Group animal # (D14) (D 21) (D 28) (D 35) (D 42) (D 56) (D 70) 1 ug 666 346 537 488 420339 383 5106 vA317/ 667 589 633 608 777 730 480 5340 RV01 641 577 969986 1286 673 460 5241 (29) 669 385 760 562 409 386 251 2742 670 218 222266 184 168 105 1520 GMT 397 561 535 501 405 295 3589 1 ug 671 292 319307 305 186 171 1276 vA317/ 672 141 152 194 200 91 43 2379 CNE17 673 161516 440 389 236 150 5264 674 239 499 455 422 281 162 4442 675 292 240157 129 72 43 2524 GMT 215 313 285 265 152 95 2823

TABLE III-7 RSV serum neutralization titers of cotton rats, 5 animalsper group, after intramuscular vaccination on days 0 and 56 with theindicated vaccines. Serum was collected for antibody analysis on days 14(2wp1), 21 (3wp1), 28 (4wp1), 35 (5wp1), 42 (6wp1), 56 (8wp1) and 70(2wp2). Data are represented as 60% RSV neutralization titers for 1pools of 5 animals per group. If an individual pool had a titer of <20(limit of detection) it was assigned a titer of 10. RSV serumneutralization titers 2wp1 3wp1 4wp1 5wp1 6wp1 8wp1 2wp2 Group (D 14) (D21) (D 28) (D 35) (D 42) (D 56) (D 70) 1 ug vA317/RV01 52 82 90 106 80101 1348 (29) 1 ug vA317/CNE17 34 38 36 33 25 36 143

Example IV—Sequential Administration of RNA and Protein to Rats (Study2)

The vA142 replicon, which expresses the full-length wild type surfacefusion glycoprotein of RSV with the fusion peptide deleted, was used forthis experiment. Cotton rats, 4-8 animals per group, were givenintramuscular vaccinations (100 μL in one leg) on days 0 and 21. Allcotton rats in this study (except naive) were vaccinated with 5 μg Fsubunit+alum on day 49 (four weeks after the second vaccination).

Group 1 self-replicating RNA (vA142, 1 μg, RSV-F) formulated in LNPs(RV01(37). The LNP had the following composition: 40% DlinDMA, 10% DSPC,48% Chol, 2% PEG DMG 2000 and an N:P ratio of 8:1. They were made usingMethod B, except a 175 μg RNA batch size was used.

Group 2 self-replicating RNA (vA142, 0.1 μg, RSV-F) formulated in LNPs(RV01(37). The LNP had the following composition: 40% DlinDMA, 10% DSPC,48% Chol, 2% PEG DMG 2000 and an N:P ratio of 8:1. They were made usingMethod B, except a 175 μg RNA batch size was used.

Group 3 self-replicating RNA (vA142, 1 μg, RSV-F) formulated in LNPs(RV17(10). The LNP had the following composition: NVP-LJC305-NX-2 40%,DSPC—10%, Chol—49.5%, PEG DMG 5000-0.5% and an N:P ratio of 8:1. Theywere made using Method A, except a 200 μg RNA batch size was used.

Group 4 self-replicating RNA (vA142, 0.1 μg, RSV-F) formulated in LNPs(RV17(10). The LNP had the following composition: NVP-LJC305-NX-2 40%,DSPC-10%, Chol—49.5%, PEG DMG 5000-0.5% and an N:P ratio of 8:1. Theywere made using Method A, except a 200 μg RNA batch size was used.

Group 5 self-replicating RNA (vA142, 1 μg, RSV-F) formulated in LNPs(RV05(11). The LNP had the following composition: NVP-LGB046-NX-1 40%,18:2 PE (DLoPE)—30%, Chol—28%, PEG DMG 2000-2% and an N:P ratio of 8:1.They were made using Method A, except a 200 μg RNA batch size was used.

Group 6 self-replicating RNA (vA142, 0.1 μg, RSV-F) formulated in LNPs(RV05(11). The LNP had the following composition: NVP-LGB046-NX-1 40%,18:2 PE (DLoPE)—30%, Chol—28%, PEG DMG 2000-2% and an N:P ratio of 8:1.They were made using Method A, except a 200 μg RNA batch size was used.

Group 7 self-replicating RNA (vA142, 1.0 μg, RSV-F) formulated withCNE13 at an N:P ratio of 10:1.

Group 8 self-replicating RNA (vA142, 0.1 μg, RSV-F) formulated withCNE13 at an N:P ratio of 10:1.

Group 9 self-replicating RNA (vA142, 1.0 μg, RSV-F) formulated withCNE17 at an N:P ratio of 10:1.

Group 10 self-replicating RNA (vA142, 0.1 μg, RSV-F) formulated withCNE17 at an N:P ratio of 10:1.

Group 11 VRPs (1×10⁶ IU) expressing the full-length wild type surfacefusion glycoprotein of RSV.

Group 12 RSV-F subunit protein vaccine (5 μg) adjuvanted with alum.

Group 13 a naïve control (3 animals).

Serum was collected for antibody analysis on days 0, 21, 35, 49, 64.

Results

F-specific serum IgG titers are shown in table IV-1 (day 21), IV-2 (day35), IV-3 (day 49), IV-4 (day 64). RSV serum neutralizing antibodytiters on days 21, 35, 49 and 64 are shown in table IV-5.

Conclusions

In this study, cotton rats were vaccinated with vA142 repliconformulated RV01, RV17, RV05, CNE17, or CNE13. Each of these vaccines wasgiven at two doses (1.0 and 0.1 μg). After the first repliconvaccination, F-specific serum IgG titers were highest with RV01,followed by RV05, RV17 and CNE17 (40-67% of the F-specific IgG titerinduced by a matched dose of vA142+RV01), and finally CNE13 (6-10% ofthe F-specific IgG titer induced by a matched dose of vA142+RV01). Afterthe first replicon vaccination, RSV neutralization titers wereapproximately equal for RV01, RV05, RV17 and CNE17, whereas titers withCNE13 were lower (35-68% of the RSV neutralization titer induced by amatched dose of vA142+RV01). Titers in all groups were boosted by ahomologous second vaccination given on day 21. After the second repliconvaccination, F-specific serum IgG titers were again highest with RV01,followed closely by RV05, RV17, and CNE17, and lower with CNE13 (25-33%of the F-specific IgG titer induced by a matched dose of vA142+RV01).Post second vaccination RSV neutralization titers generally followedthis same trend, although exhibited more variability within the groups.RSV serum neutralization titers two weeks after the second vaccinationwere at least three fold lower in replicon groups than F subunit+alum orVRP groups.

All cotton rats in this study (except naive) were vaccinated with 5 μg Fsubunit+alum on day 49 (four weeks after the second vaccination). Thisthird vaccination did not boost antibody titers in cotton ratspreviously vaccinated with subunit, but it provided a large boost totiters in cotton rats previously vaccinated with replicon, regardless ofthe formulation (RV01, RV17, RV05, CNE17, CNE13). In most cases the RSVserum neutralization titers after two replicon vaccinations followed byan F subunit+alum boost were equal to titers induced by two or threesequential F subunit+alum vaccinations.

For each group and time point a ratio of F-specific IgG titer to RSVneutralizing titer can be calculated, and is one indication of thequality of the antibody response. A lower ratio suggests that a largerfraction of the vaccine-induced antibody is capable of neutralizing RSV,and is therefore functional. The data from this study show that theF-specific IgG:RSV neutralizing antibody titer ratio is lower aftervaccination with replicon or VRP than after vaccination with Fsubunit+alum. The lower F-specific IgG:RSV neutralizing antibody titerratio set by two replicon or VRP vaccinations was maintained after a Fsubunit+alum boost.

TABLE IV-1 F-specific serum IgG titers of cotton rats, 3-8 animals pergroup, 21 days after intramuscular vaccination with the indicatedvaccines. Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <25 (limit of detection) it was assigned a titer of 5. Group 3wp1(day 21) F-specific serum IgG titers GMT 1 ug vA142/RV01 (37) 334 316943 628 787 550 546 639 558 0.1 ug vA142/RV01 (37) 193 90 251 66 30 8289 388 112 1 ug vA142/RV17 (10) 861 131 378 262 736 200 327 265 330 0.1ug vA142/RV17 (10) 188 140 5 41 111 28 51 1 ug vA142/RV05 (11) 376 630370 383 232 126 551 342 0.1 ug vA142/RV05 (11) 93 192 38 181 5 57 5 20249 1 ug vA142/CNE13 5 185 681 268 192 455 415 441 201 0.1 ug vA142/CNE13164 49 5 66 44 243 207 184 76 1 ug vA142/CNE17 43 214 46 5 5 33 48 12935 0.1 ug vA142/CNE17 5 5 5 5 53 186 5 5 11 1E6 VRP (F-full) 1624 11013111 3952 1944 1097 573 1271 1555 5 ug F trimer subunit/alum 1249 48824687 17552 29911 22928 16624 8425 naive 5 5 5 5

TABLE IV-2 F-specific serum IgG titers of cotton rats, 3-8 animals pergroup, after intramuscular vaccination with the indicated vaccines ondays 0 and 21. Serum was collected for antibody analysis on day 35(2wp2). Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <25 (limit of detection) it was assigned a titer of 5. Group 2wp2(day 35) F-specific serum IgG titers GMT 1 ug vA142/RV01 3608 1966 42202961 3896 4212 14834 2680 3938 (37) 0.1 ug vA142/RV01 1454 412 2127 9341138 2750 2184 1839 1403 (37) 1 ug vA142/RV17 3488 1892 3568 1915 30941507 7845 3266 2927 (10) 0.1 ug vA142/RV17 599 1153 177 614 850 254 503(10) 1 ug vA142/RV05 2735 2030 3331 4957 6336 1957 3067 3207 (11) 0.1 ugvA142/RV05 1833 1490 1997 584 1307 1328 82 2348 1008 (11) 1 ugvA142/CNE13 1971 3234 15961 1679 3006 4928 1850 2366 3203 0.1 ugvA142/CNE13 3380 1879 1348 4032 2423 1902 1311 2583 2195 1 ugvA142/CNE17 241 3321 838 522 452 986 1646 2759 958 0.1 ug vA142/CNE17171 997 419 267 225 1270 368 983 459 1E6 VRP (F-full) 4944 5023 560312192 14184 5764 6392 10678 7448 5 ug F trimer 60431 34850 94259 11620375778 108013 124309 81297 subunit/alum naive 5 5 5 5

TABLE IV-3 F-specific serum IgG titers of cotton rats, 3-8 animals pergroup, after intramuscular vaccination with the indicated vaccines ondays 0 and 21. Serum was collected for antibody analysis on day 49(4wp2). Data are represented as titers for individual animals and thegeometric mean titer for each group. If an individual animal had a titerof <25 (limit of detection) it was assigned a titer of 5. Group 4wp2(day 49) F-specific serum IgG titers GMT 1 ug vA142/RV01 1681 961 21542220 2861 2710 9271 1870 2383 (37) 0.1 ug vA142/RV01 1271 254 935 671870 1446 1485 1650 943 (37) 1 ug vA142/RV17 3595 1977 2836 1408 43141075 2652 1811 2239 (10) 0.1 ug vA142/RV17 745 787 137 468 752 570 503(10) 1 ug vA142/RV05 2309 1028 1957 3162 2916 2343 2123 2151 (11) 0.1 ugvA142/RV05 1899 897 1234 855 482 488 5 2268 513 (11) 1 ug vA142/CNE13922 2475 7883 964 1626 3663 1241 1124 1861 0.1 ug vA142/CNE13 1702 12341090 2190 1261 1066 878 1766 1341 1 ug vA142/CNE17 238 6470 884 879 5301066 1186 4942 1188 0.1 ug vA142/CNE17 237 613 211 186 158 744 301 715331 1E6 VRP (F-full) 2537 2594 1662 6465 10802 3442 3796 6875 4023 5 ugF trimer 36694 18062 65268 62018 55760 89468 110556 54776 subunit/alumnaive 5 5 5 5

TABLE IV-4 F-specific serum IgG titers of cotton rats, 3-8 animals pergroup, after intramuscular vaccination on days 0 and 21 with theindicated vaccines. All cotton rats (except those in naive group) werealso vaccinated with 5 μg of F trimer subunit/alum on day 49 (4wp2).Serum was collected for antibody analysis on day 64 (~2wp3). Data arerepresented as titers for individual animals and the geometric meantiter for each group. If an individual animal had a titer of <25 (limitof detection) it was assigned a titer of 5. Group ~2wp3 (day 64)F-specific serum IgG titers GMT 1 ug vA142/RV01 (37) 15772 6101 1478016995 17241 25359 27454 19522 16563 0.1 ug vA142/RV01 25676 13765 1959711461 7832 16681 12694 20780 15123 (37) 1 ug vA142/RV17 (10) 20604 2242656417 29296 53866 14992 19221 17081 25900 0.1 ug vA142/RV17 20857 240829670 15857 19641 53866 20821 (10) 1 ug vA142/RV05 (11) 32193 23171 2241628063 19311 34924 16713 24494 0.1 ug vA142/RV05 38208 25998 17304 2063414551 19200 1528 19917 15308 (11) 1 ug vA142/CNE13 10884 15381 5243618745 22023 20262 24805 25415 21537 0.1 ug vA142/CNE13 17474 35952 3739323943 39407 16807 17189 54958 27753 1 ug vA142/CNE17 7543 77979 428897306 14673 28679 37562 54571 25128 0.1 ug vA142/CNE17 21964 22163 3586033383 23572 17133 39531 110957 31748 1E6 VRP (F-full) 17455 27475 1586620471 34132 29351 22155 56379 25777 5 ug F trimer 83936 38397 10029797227 84404 63790 159161 82911 subunit/alum naive 5 5 5 5

TABLE IV-5 RSV serum neutralization titers of cotton rats, 2-8 animalsper group (2 per group for naive) after intramuscular vaccinations withthe indicated vaccines on days 0 and 21 (3wp1). All animals (exceptnaive) were then vaccinated with 5 μg F trimer subunit/alum on day 49(4wp2). Serum was collected for analysis on days 21 (3wp1), 35 (2wp2),49 (4wp2), and 64 (~2wp3). Data are represented as 60% RSVneutralization titers for 2 pools of 3-4 animals per group and geometricmean titers of these 2 pools per group (one pool of 3 animals for naivegroup). If an individual pool had a titer of <20 (limit of detection) itwas assigned a titer of 10. RSV serum neutralization titers 3wp1 (D 21)2wp2 (D 35) 4wp2 (D 49) ~2wp3 (D 64) Group pool 1 pool 2 GMT pool 1 pool2 GMT pool 1 pool 2 GMT pool 1 pool 2 GMT 1 ug vA142/RV01 61 72 66 5251184 788 230 407 306 2742 2580 161 (37) 0.1 ug vA142/RV01 26 26 26 105251 162 33 101 58 887 3542 1772 (37) 1 ug vA142/RV17 87 54 69 301 281291 195 201 198 2177 3266 3221 (10) 0.1 ug vA142/RV17 26 23 24 128 40 7249 38 43 2219 581 1135 (10) 1 ug vA142/RV05 70 80 75 468 428 448 178 228201 3962 8295 5733 (11) 0.1 ug vA142/RV05 29 25 27 610 226 371 192 138163 3893 1541 2449 (11) 1 ug vA142/CNE13 32 54 42 358 270 311 181 113143 709 1987 1187 0.1 ug vA142/CNE13 38 39 38 523 118 248 77 131 1002080 1553 1797 1 ug vA142/CNE17 21 25 23 568 142 284 420 156 256 44446149 5227 0.1 ug vA142/CNE17 10 31 18 65 56 60 37 52 44 3040 5984 42651E6 VRP (F-full) 172 109 137 2472 3354 2879 734 1442 1029 1768 2085 19205 ug F trimer 248 379 307 2031 3252 2570 607 2081 1124 2720 3085 2897subunit/alum naive 10 10 10 10

Example V—Sequential Administration of RNA and Protein to Cattle

The vA317 replicon that expresses the surface fusion glycoprotein of RSV(RSV-F) was used for this experiment. Calves (5 per group) were givenintramuscular vaccinations on days 0, 21, 86 and 146. RNA and PBSvaccines were administered as 1.0 mL split doses on each side of theneck (2 mL total dose). The Triangle 4 product (a vaccine for preventionof disease caused by bovine respiratory syncytial virus as well as otherpathogens; Ft. Dodge Animal Health) was administered as labeled (2.0 mLdose on one side of the neck). The RSV-F subunit protein vaccine (15 μg)adjuvanted with MF59 was administered as 1.0 mL split doses on each sideof the neck (2 mL total dose). Self-replicating RNA (vA317, 60 μg) wasformulated in LNP (RV01(01), with the following composition: 40%DlinDMA, 10% DSPC, 48% Chol, 2% PEG DMG 2000, N:P ratio of 8:1 and wasmade using Method C, except a 1.5 mg RNA batch size was used. Selfreplicating RNA (vA317, 60 μg) was formulated with CNE17 at an N:P ratioof 10:1.

Calves were vaccinated three times with RNA/CNE, RNA/LNP, a commercialRSV vaccine (Triangle 4), or PBS. It should be noted that the RNAvaccines in this study encoded human RSV F while the Triangle 4contained bovine RSV F (the RSV F protein is highly conserved betweenBRSV and HRSV). All calves were vaccinated a fourth time with RSV-Fsubunit/MF59. Serum was collected for antibody analysis on days 0, 14,21, 35, 42, 56, 63, 86, 100, 107, 114, 121, 128, 135, 146, 160, 167,174, 181, 188, 195, and 202.

Results

F-specific serum IgG titers on days 21, 35, 56, 86, 100, 121, 146, 160,181 and 202 are shown in table V-1. RSV serum neutralizing antibodytiters on days 35, 56, 100, 107, 114, 146, 160, 167, 174 are shown intable V-2.

Conclusions

The data from this study provided proof of concept for RNA replicon RSVvaccines in large animals, with two of the five RNA/CNE-vaccinatedcalves demonstrating good neutralizing antibody titers after the thirdvaccination as measured by the complement-independent HRSVneutralization assay. In the complement-enhanced HRSV neutralizationassay, all vaccinated calves had good neutralizing antibody titers afterthe second RNA vaccination regardless of the formulation. Furthermore,both RNA/CNE and RNA/LNP elicited F-specific serum IgG titers that weredetected in a few calves after the second vaccination and in all calvesafter the third vaccination. Proof of concept for RNA replicon vaccinesin large animals is particularly important in light of the loss inpotency observed previously with DNA-based vaccines when moving fromsmall animal models to larger animals and humans. A typical dose for DNAin cows is 0.5-1.0 mg (Taylor 2005, Boxus 2007), so it is veryencouraging that immune responses were induced with these 1^(st)generation RNA vaccines at a 60 μg dose. The RNA/LNP vaccine was alsoshown to be superior to DNA in the mouse model experiments.

These data also suggest that CNE-formulated RNA is more immunogenic thanthe LNP-formulated RNA in calves. This was evident in higher ELISAtiters after the second vaccination, and higher neutralization titersafter the third vaccination with the former formulation. RSV-Fsubunit/MF59 was able to boost the IgG response in all previouslyvaccinated calves, and boost complement-independent HRSV neutralizationtiters of calves previously vaccinated with RNA.

TABLE V-1 F-specific serum IgG titers of cows, 5 animals per group,after intramuscular vaccinations with the indicated vaccines on days 0,21, 86. All cows were vaccinated with 15 μg of F trimer subunit + MF59on day 146. Serum was collected for antibody analysis prior tovaccination (pre-immune) and on days 21, 35, 56, 86, 100, 121, 146, 160,181, and 202. Data are represented as titers for individual cows and thegeometric mean titers of 5 individual cows per group. If an individualanimal had a titer of <25 (limit of detection) it was assigned a titerof 5. F-specific serum IgG titers pre- 3wp1 2wp2 5wp2 ~9wp2 2wp3 5wp37wp3 2wp4 5wp4 8wp4 Group animal # immune D 21 D 35 D 56 D 86 D 100 D121 D 146 D 160 D 181 D 202 PBS 2 5 5 5 5 5  5  5  5   36  49  139 5 5 55 5 5  5  5  5   76  179  154 6 5 5 5 5 5  5  5  5  331  368  322 11 5 55 5 5  5 20 5 5 5 5 5  5  5  5   5  29  73 GMT 5 5 5 5 5  5  5  5   46 98  150 60 ug 1 5 5 62 36 28 4182  1518  193 141475  28402  6864 vA317/7 5 5 5 5 5 510 328  32 11228 4560 1437 RV01 8 5 5 34 5 5 133 223  9811078 3637 1228 (01) 10 5 5 5 30 86 1061  13 5 5 5 5 49 887 301  5110583 5162 2531 GMT 5 5 12 11 20 768 428  74 20774 7022 2353 60 ug 14 55 5 5 5 191 177  5  4609 2093 1121 vA317/ 17 5 5 1561 828 610 1730 1785  110  6357 2912 1611 CNE17 19 5 5 243 167 228 3239  1604  411 8628020052  10056  22 5 5 5 58 155 278 216 132 16082 8084  811 23 5 5 5 5 5927 410  55  967 2697 2601 GMT 5 5 34 46 56 773 538  70  8297 4843 2073Triangle 3 5 5 4014 3242 2920 24028  18303  961 25068 14047  4876 4 9 55 930 715 686 2552  7514  325 12088 4099 2143 15 5 5 1274 373 145 1363 1095  140  8976 2814 1673 18 5 5 413 97 109  74* 1058*  109*  37419*11207*  4461* 24 5 5 9200 2319 1138 1611  400 292 11771 3209 1184 GMT 55 1784 721 514 3406  2786  336 13376 4775 2133 *Cow #18 was vaccinatedwith vA317/CNE17 vaccine at D 86 (instead of Triangle 4), and thereforeits titers are not included in the GMT from day 100 onwards.

TABLE V-2 RSV serum neutralization titers of cows, 5 animals per group,after intramuscular vaccinations with the indicated vaccines on days 0,21, 86. All cows were vaccinated with 15 μg of F trimer subunit + MF59on day 146. Serum was collected for analysis prior to vaccination(pre-immune) and on days 35, 56, 100, 107, 114, 146, 160, 167, and 174.Data are represented as 60% RSV neutralization titers of individual cowsand the geometric mean titers of 5 individual cows per group. If anindividual animal had a titer of <20 (limit of detection) it wasassigned a titer of 10. RSV serum neutralization titers pre- 2wp2 5wp22wp3 3wp3 4wp3 8wp3 2wp4 3wp4 4wp4 Group animal # immune D 35 D 56 D 100D 107 D 114 D 146 D 160 D 167 D 174 PBS 2 10 10 10 27 26 10 38 10 10 105 10 10 10 23 24 21 10 10 10 10 6 27 10 10 10 32 27 10 10 10 10 11 10 1010 10 10 22 20 10 10 10 10 10 24 10 10 10 10 GMT 12 10 10 14 18 20 14 1010 10 60 ug 1 31 10 10 30 35 39 10 310  156  78 vA317/ 7 10 10 10 21 1035 10 25 10 10 RV01 8 10 10 10 22 10 10 10 29 28 10 (01) 10 10 10 10 1010 10 13 10 10 10 26 10 10 27 21 10 27 GMT 13 10 10 20 13 17 13 47 26 2160 ug 14 10 10 10 10 10 21 10 10 10 10 vA317/ 17 10 10 10 99 93 123  10145  110  75 CNE17 19 10 10 10 173 179  132  48 420  379  233  22 10 1032 10 35 39 10 82 52 58 23 10 10 10 10 28 28 10 22 28 10 GMT 10 10 13 2844 52 14 64 57 40 Triangle 3 10 30 39 49 46 39 27 31 24 21 4 9 21 10 1042 33 60 10 10 67 23 15 10 10 10 23 30 34 10 37 27 10 18 10 10 10 10 60*  62*  10*  67*  70*  62* 24 10 27 10 51 45 35 10 30 10 10 GMT 12 1513 39 38 41 13 24 26 15 *Cow #18 was vaccinated with vA317/CNE17 vaccineat D 86 (instead of Triangle 4), and therefore its titers are notincluded in the GMT from day 100 onwards.

Example VI—Alphavirus VRP Prime, Protein Boost and Coadministration ofRNA and Subunit

Mice were immunized three times, three weeks apart with VRPs expressingthe gH/gL complex, VRPs expressing green fluorescent protein (VRP-G),purified gH/gL subunit with or without MF59, different sequences of VRPor VRP-G followed by subunit in PBS or MF59, VRPs expressing the gH/gLcomplex mixed with purified gH/gL subunit, or VRP-G mixed with purifiedgH/gL subunit (see Table VI-1). A group of control mice did not receiveany vaccine, and additional control groups included GFP VRPs todetermine whether any effects from VRP prime were specific to theencoded antigen.

TABLE VI-1 Group Mice Vaccinations Antigen Formulation Dose 1 4 3 None —— 2 8 3 gH FL/gL VRP — 10⁶ IU 3 8 3 gHsol/gL subunit PBS 1 μg 4 8 3gHsol/gL subunit MF59 1 μg 5 8 3 1st dose VEE gH FL/gL VRP — 10⁶ IU 2ndand 3rd dose gHsol/gL subunit MF59 1 μg 6 8 3 1st and 2nd doses VEE gHFL/gL VRP — 10⁶ IU 3rd dose gHsol/gL subunit MF59 1 μg 7 8 3 1st doseVEE GFP VRP (VRP-G) — 10⁶ IU 2nd and 3rd dose gHsol/gL subunit MF59 1 μg8 8 3 1st and 2nd doses VRP-G — 10⁶ IU 3rd dose gHsol/gL subunit MF59 1μg 9 8 3 1st dose gH FL/gL VRP — 10⁶ IU 2nd and 3rd dose gHsol/gLsubunit PBS 1 μg 10 8 3 1st and 2nd doses gH FL/gL VRP — 10⁶ IU 3rd dosegHsol/gL subunit PBS 1 μg 11 8 3 gH FL/gL VRP + gHsol/gL — 10⁶ IU + 1 μgsubunit MIXED 12 8 3 GFP VRP + gHsol/gL subunit — 10⁶ IU + 1 μg MIXED

Sera were harvested three weeks after each immunization and used forELISA to determine binding antibody titers, using the same purifiedgH/gL antigen in the assay as in the subunit vaccine. The sera were alsoused for HCMV microneutralization assay using TB40 infection of ARPE-19epithelial cells.

Four weeks after the third immunization, spleens were extracted fromsacrificed mice. Spleen cells were stimulated in vitro with purifiedgH/gL protein or a pool of 15-mer peptides (overlapping by 11 aminoacids) corresponding approximately to the c-terminal half of the gHprotein, stained for CD4, CD8, and cytokine expression, and analyzedusing flow cytometry.

Vaccination with the gH/gL VRPs elicited strongly neutralizing antibodyresponses (FIG. 1). Purified gH/gL subunit elicited modest responseswithout adjuvant. When formulated with MF59 potently neutralizingantibody responses that were at least as strong as those elicited bygH/gL VRPs were elicted. Different VRP prime, subunit/MF59 boostregimens did not increase the neutralizing titer compared to subunit.Substituting the gH/gL VRP for a GFP-expressing GFP in the prime-boostregimens had the expected result of decreasing the magnitude of theneutralizing response. VRP prime followed by unadjuvanted protein boostdid not increase neutralizing titers compared to subunit/MF59 alone. Thetiters for any given prime boost regimen were comparable when thesubunit boost was delivered with or without MF59.

Vaccination with VRPs or subunit without adjuvant elicited weak CD4+ Tcell responses (FIG. 2). Subunit adjuvanted with MF59 elicited CD4 cellswith a Th2/Th0 phenotype. The only combination of VRPs and subunit(prime-boost or mixed modality) that elicited significantly more CD4cells than subunit/MF59 was one dose of VRPs followed by two doses ofsubunit/MF59. These cells had a Th1/Th0 phenotype; all groups thatreceived a gH/gL-expressing VRP elicited cells with this phenotype. Incontrast, priming or co-immunizing with GFP-expressing VRPs did notchange the T cell phenotype compared to subunit/MF59.

Vaccination with VRPs expressing gH/gL elicited robust CD8+ T cellresponses (FIG. 3). No prime boost combination tested orco-administration with subunit improved the responses.

The data from this study showed gH/gL prime followed by gH/gL subunitboost elicited strong neutralizing titers with a concomitant CD4 T cellresponse, which may affect durability of the responses. As a result,following VRP prime, there is no need to adjuvant the subunit boost withMF59. VRPs that expressed a heterologous antigen did not prime animalsfor subsequent subunit boost, and did not adjuvant responses to subunitwhen co-delivered.

Example VII—A SAM™ Vaccine Prime, Protein Boost and Coadministration ofRNA and Subunit Using the HCMV Pentameric Antigen

Mice were immunized three times, three weeks apart with a SAM vaccine,which is a self replicating RNA as described herein, encoding the CMVpentameric complex (gH/gL/UL128/UL130/UL131), purified pentamericsubunit adjuvanted with MF59, different sequences of SAM followed bysubunit in MF59, or a combination of the two (Table VII-1). The SAMvaccine was encapsulated in synthetic LNPs for non-viral delivery. Agroup of control mice did not receive any vaccine.

TABLE VII-1 No. No. Group Mice Doses Antigen Formulation Dose 1 4 — — —1 2 8 3 SAM vaccine encoding Lipid 1 microgram pentameric complex (Pentananoparticle SAM) (LNP) 3 8 3 Purified pentameric complex MF59 1microgram (Penta subunit) 4 8 3 1st Penta SAM vaccine LNP 1 microgram2nd and 3rd Penta subunit MF59 5 8 3 1st and 2nd Penta SAM LNP 1microgram vaccine 3rd Penta subunit MF59 6 8 3 Penta SAM vaccine + PentaLNP 1 microgram + subunit (mixed) 1 microgram

Sera were harvested three weeks alter each immunization and used forELISA to determine binding antibody titers, using the same purifiedantigen in the assay as in the subunit vaccine. The sera were also usedfor HCMV microneutralization assay using TB40 or VR1814 infection ofARPE-19 epithelial cells. Three or four weeks after the thirdimmunization, spleens were extracted from sacrificed mice. Spleen cellswere stimulated in vitro with purified protein or a pool of 15-merpeptides (overlapping by 11 amino acids) corresponding to the c-terminalhalf of the gH protein, stained for cytokine expression, and analyzedusing flow cytometry.

The SAM vaccine and subunit/MF59 alone elicited potently neutralizingantibody responses after three doses (FIG. 4). The pentameric subunit inMF59 did not respond as well as pentameric SAM vaccine to the first andsecond dose of vaccine, but titers elicited by subunit/MF59 surpassedtiters elicited by SAM vaccine after the third dose. One SAM vaccineprime followed by a single dose of subunit/MF59 elicited strongerneutralizing responses than two doses of subunit/MF59, but was equal toSAM vaccine alone. A second subunit/MF59 boost administered to theseanimals raised neutralizing responses to a level that exceeded thoseseen after three doses of subunit/MF59 or SAM vaccine. Two doses of SAMvaccine followed by a single dose of subunit/MF59 did not appear tobenefit neutralizing responses compared to either subunit/MF or SAMvaccine alone. Mixing SAM vaccine with subunit, without MF59, elicited astrong response after the first dose, similar to RNA alone, and elicitedstrong neutralizing titers after two and three doses.

CD4+ T cell responses to the vaccinations using purified gH/gL andpentameric subunits were analyzed. SAM vaccine prime protein boost andmixed SAM vaccine+subunit elicited more CD4+ T cells responding to gH/gLre-stimulation than SAM vaccine or subunit/MF59 alone (FIG. 5). CD4+responses to RNA alone were insignificant, whereas responses tosubunit/MF59 alone were Th2/Th0 phenotype. The phenotype of theresponding cells from mice immunized combinations of SAM vaccine andsubunit was primarily Th1/Th0. Similar trends were seen whenre-stimulating cells with purified pentameric complex, although theresponses were generally stronger (FIG. 6).

CD8+ T cell responses to the vaccinations using purified pentamericsubunit or a pool of peptides to gH were also analyzed. The onlysignificant CD8 responses seen when re-stimulating with pentamericsubunit was in the mice immunized with two doses of SAM vaccine followedby one dose of subunit/MF59 (FIG. 7). Cells from these animals alsoshowed the strongest responses when re-stimulated with gH peptides (FIG.8). Mice immunized with the SAM vaccine+subunit, with one dose of SAMvaccine followed by two doses of subunit/MF59, or with SAM vaccinealone, also showed significant responses to re-stimulation (FIG. 8).

Conclusions: One dose of SAM vaccine followed by two doses ofsubunit/MF59, as well as SAM vaccine+subunit, elicited higherneutralizing titers than subunit/MF59 alone. The response to SAMvaccine+subunit did not require addition of MF59 adjuvant. The largestimpact of SAM vaccine prime subunit/MF59 boost was on cellular immuneresponses. Any combination including the SAM vaccine produced primarilya Th1/Th0 CD4+ response. Moreover, two immunizations with SAM vaccinefollowed by one immunization with subunit/MF59 produced the strongestCD8+ responses. This study shows that a SAM vaccine prime protein boostregimen can be optimized to produce a desired immune response, i.e.cellular or humoral.

Example VIII—RSV F RNA Prime (Dose Range), Protein Boost (3 μg, VaryingFormulation)

RSV-F RNA prime (as a dose range), and a subunit boost (3 μg, differentformulations) were administered to mice to evaluate the ability ofsubunit, with different formulations as indicated in Table VIII-1, toboost RNA-induced response. Each mouse received a constant 3 μg subunitdose, with variable RNA doses and different subunit adjuvants. Mice werevaccinated on days 0, 21 and 42 intramuscularly with a 100 μl dose,split between the two hind legs. Serum was sampled on day 20 forF-specific IgG, and on days 35 and 56 for IgG, IgG1, IgG2a andneutralizing antibody. Spleens were harvested on day 56.

TABLE VIII-1 Group n 1st & 2nd Vacc. 3rd Vacc. 1 7 1 μg RNA/CMF34 3 μg F2 7 0.01 μg RNA/CMF34 3 7 0.3 ng RNA/CMF34 4 7 3 μg F 5 7 1 μg RNA/CMF343 μg F/alum 6 7 0.01 μg RNA/CMF34 7 7 0.3 ng RNA/CMF34 8 7 3 μg F/alum 97 1 μg RNA/CMF34 3 μg F/MF59 10 7 0.01 μg RNA/CMF34 11 7 0.3 ngRNA/CMF34 12 7 3 μg F/MF59 13 7 1 μg RNA/CMF34 (only 2nd) 1 μg RNA/CMF34(T cell ctrl.) 14 7 0.01 μg RNA/CMF34 (only 2nd) 0.1 μg RNA/CMF34 (Tcell ctrl) 15 7 0.3 ng RNA/CMF34 (only 2nd) 0.3 ng RNA/CMF34 (T cellctrl) 16 7 PBS PBS

The data showed that RNA prime, subunit boost is an effective regimenfor inducing high titer of RSV neutralizing antibodies (FIG. 9). Theboost works with alum-adjuvanted, MF59-adjuvanted, or even unadjuvantedF subunit boost (FIG. 11).

The RNA vaccine set a Th1-biased response (IgG2a, Th1 cytokines) thatwas maintained after the subunit boost, irrespective of the boostadjuvant, and even when the subunit with or without adjuvant vaccineinduced more of a Th2-biased response on its own (without an RNA prime).Very low doses of the RNA vaccine, even those without a detectableimmune response, were sufficient to set the Th1-biased response that wasmaintained after the subunit boost. (FIG. 10)

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments.

SEQUENCES CMV gB FL (SEQ ID NO: 1): 1 -atggaaagccggatctggtgcctggtcgtgtgcgtgaacctgtgcatcgtgtgcctgggagccgccgtgagcagcagcagcaccagaggcaccagcgccacacacagccaccacagcagccacaccacctctgccgcccacagcagatccggcagcgtgtcccagagagtgaccagcagccagaccgtgtcccacggcgtgaacgagacaatctacaacaccaccctgaagtacggcgacgtcgtgggcgtgaataccaccaagtacccctacagagtgtgcagcatggcccagggcaccgacctgatcagattcgagcggaacatcgtgtgcaccagcatgaagcccatcaacgaggacctggacgagggcatcatggtggtgtacaagagaaacatcgtggcccacaccttcaaagtgcgggtgtaccagaaggtgctgaccttccggcggagctacgcctacatccacaccacatacctgctgggcagcaacaccgagtacgtggcccctcccatgtgggagatccaccacatcaacagccacagccagtgctacagcagctacagccgcgtgatcgccggcacagtgttcgtggcctaccaccgggacagctacgagaacaagaccatgcagctgatgcccgacgactacagcaacacccacagcaccagatacgtgaccgtgaaggaccagtggcacagcagaggcagcacctggctgtaccgggagacatgcaacctgaactgcatggtcaccatcaccaccgccagaagcaagtacccttaccacttcttcgccacctccaccggcgacgtggtggacatcagccccttctacaacggcaccaaccggaacgccagctacttcggcgagaacgccgacaagttcttcatcttccccaactacaccatcgtgtccgacttcggcagacccaacagcgctctggaaacccacagactggtggcctttctggaacgggccgacagcgtgatcagctgggacatccaggacgagaagaacgtgacctgccagctgaccttctgggaggcctctgagagaaccatcagaagcgaggccgaggacagctaccacttcagcagcgccaagatgaccgccaccttcctgagcaagaaacaggaagtgaacatgagcgactccgccctggactgcgtgagggacgaggccatcaacaagctgcagcagatcttcaacaccagctacaaccagacctacgagaagtatggcaatgtgtccgtgttcgagacaacaggcggcctggtggtgttctggcagggcatcaagcagaaaagcctggtggagctggaacggctcgccaaccggtccagcctgaacctgacccacaaccggaccaagcggagcaccgacggcaacaacgcaacccacctgtccaacatggaaagcgtgcacaacctggtgtacgcacagctgcagttcacctacgacaccctgcggggctacatcaacagagccctggcccagatcgccgaggcttggtgcgtggaccagcggcggaccctggaagtgttcaaagagctgtccaagatcaaccccagcgccatcctgagcgccatctacaacaagcctatcgccgccagattcatgggcgacgtgctgggcctggccagctgcgtgaccatcaaccagaccagcgtgaaggtgctgcgggacatgaacgtgaaagagagcccaggccgctgctactccagacccgtggtcatcttcaacttcgccaacagctcctacgtgcagtacggccagctgggcgaggacaacgagatcctgctggggaaccaccggaccgaggaatgccagctgcccagcctgaagatctttatcgccggcaacagcgcctacgagtatgtggactacctgttcaagcggatgatcgacctgagcagcatctccaccgtggacagcatgatcgccctggacatcgaccccctggaaaacaccgacttccgggtgctggaactgtacagccagaaagagctgcggagcagcaacgtgttcgacctggaagagatcatgcgggagttcaacagctacaagcagcgcgtgaaatacgtggaggacaaggtggtggaccccctgcctccttacctgaagggcctggacgacctgatgagcggactgggcgctgccggaaaagccgtgggagtggccattggagctgtgggcggagctgtggcctctgtcgtggaaggcgtcgccacctttctgaagaaccccttcggcgccttcaccatcatcctggtggccattgccgtcgtgatcatcacctacctgatctacacccggcagcggagactgtgtacccagcccctgcagaacctgttcccctacctggtgtccgccgatggcaccacagtgaccagcggctccaccaaggataccagcctgcaggccccacccagctacgaagagagcgtgtacaacagcggcagaaagggccctggccctcccagctctgatgccagcacagccgcccctccctacaccaacgagcaggcctaccagatgctgctggccctggctagactggatgccgagcagagggcccagcagaacggcaccgacagcctggatggcagaaccggcacccaggacaagggccagaagcccaacctgctggaccggctgcggcaccggaagaacggctaccggcacctgaaggacagcgacgaggaagagaacgtctgataa - 2727 CMV gB FL (SEQ ID NO: 2)MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPTNEDLDEGIMVVYKRNTVAHTFKVRVYQKVLTFARSYAYIHTTYLLGSNTEYVAPPMWETHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMFDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVADEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGIXVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATIMSNMESVHNLVYAQLQFTYDTLRGYINRALAQTAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFTAGNSAYEYVDYLFKRMIDLSSTSTVDSMIALDIDFLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDFLPPYLKGLDDLMSGLGARGKAVGVATGAVGGAVASVVEGVATFLKNPFGAFTIIINAIAVVIITYLIYTRQRRLCTQPLQNLFPYLVSADGTTVTSGSTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALARLDAEQRAQQNGTDSLDGRTGTQDKGQKPNLLDRLRHRKNGYRHLKDSDEEENV--CMV gB sol 750 (SEQ ID NO: 3): 1-atggaaagccggatctggtgcctggtcgtgtgcgtgaacctgtgcatcgtgtgcctgggagccgccgtgagcagcagcagcaccagaggcaccagcgccacacacagccaccacagcagccacaccacctctgccgcccacagcagatccggcagcgtgtcccagagagtgaccagcagccagaccgtgtcccacggcgtgaacgagacaatctacaacaccaccctgaagtacggcgacgtcgtgggcgtgaataccaccaagtacccctacagagtgtgcagcatggcccagggcaccgacctgatcagattcgagcggaacatcgtgtgcaccagcatgaagcccatcaacgaggacctggacgagggcatcatggtggtgtacaagagaaacatcgtggcccacaccttcaaagtgcgggtgtaccagaaggtgctgaccttccggcggagctacgcctacatccacaccacatacctgctgggcagcaacaccgagtacgtggcccctcccatgtgggagatccaccacatcaacagccacagccagtgctacagcagctacagccgcgtgatcgccggcacagtgttcgtggcctaccaccgggacagctacgagaacaagaccatgcagctgatgcccgacgactacagcaacacccacagcaccagatacgtgaccgtgaaggaccagtggcacagcagaggcagcacctggctgtaccgggagacatgcaacctgaactgcatggtcaccatcaccaccgccagaagcaagtacccttaccacttcttcgccacctccaccggcgacgtggtggacatcagccccttctacaacggcaccaaccggaacgccagctacttcggcgagaacgccgacaagttcttcatcttccccaactacaccatcgtgtccgacttcggcagacccaacagcgctctggaaacccacagactggtggcctttctggaacgggccgacagcgtgatcagctgggacatccaggacgagaagaacgtgacctgccagctgaccttctgggaggcctctgagagaaccatcagaagcgaggccgaggacagctaccacttcagcagcgccaagatgaccgccaccttcctgagcaagaaacaggaagtgaacatgagcgactccgccctggactgcgtgagggacgaggccatcaacaagctgcagcagatcttcaacaccagctacaaccagacctacgagaagtatggcaatgtgtccgtgttcgagacaacaggcggcctggtggtgttctggcagggcatcaagcagaaaagcctggtggagctggaacggctcgccaaccggtccagcctgaacctgacccacaaccggaccaagcggagcaccgacggcaacaacgcaacccacctgtccaacatggaaagcgtgcacaacctggtgtacgcacagctgcagttcacctacgacaccctgcggggctacatcaacagagccctggcccagatcgccgaggcttggtgcgtggaccagcggcggaccctggaagtgttcaaagagctgtccaagatcaaccccagcgccatcctgagcgccatctacaacaagcctatcgccgccagattcatgggcgacgtgctgggcctggccagctgcgtgaccatcaaccagaccagcgtgaaggtgctgcgggacatgaacgtgaaagagagcccaggccgctgctactccagacccgtggtcatcttcaacttcgccaacagctcctacgtgcagtacggccagctgggcgaggacaacgagatcctgctggggaaccaccggaccgaggaatgccagctgcccagcctgaagatctttatcgccggcaacagcgcctacgagtatgtggactacctgttcaagcggatgatcgacctgagcagcatctccaccgtggacagcatgatcgccctggacatcgaccccctggaaaacaccgacttccgggtgctggaactgtacagccagaaagagctgcggagcagcaacgtgttcgacctggaagagatcatgcgggagttcaacagctacaagcagcgcgtgaaatacgtggaggacaaggtggtggaccccctgcctccttacctgaagggcctggacgacctgatgagcggactgggcgctgccggaaaagccgtgggagtggccattggagctgtgggcggagctgtggcctctgtcgtggaaggcgtcgccacctttctgaagaactgataa - 2256 Cmv gB sol 750 (SEQ ID NO: 4)MESRIWCINVCVNLIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNETIYNTTTLKYGDVVGVNTIKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWETHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTTVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQTAEAWCVDQRRTLEVFKELSKTNPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVIRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPLPPYLKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKN--CMV gB sol 692 (SEQ ID NO: 5): 1-atggaaagccggatctggtgcctggtcgtgtgcgtgaacctgtgcatcgtgtgcctgggagccgccgtgagcagcagcagcaccagaggcaccagcgccacacacagccaccacagcagccacaccacctctgccgcccacagcagatccggcagcgtgtcccagagagtgaccagcagccagaccgtgtcccacggcgtgaacgagacaatctacaacaccaccctgaagtacggcgacgtcgtgggcgtgaataccaccaagtacccctacagagtgtgcagcatggcccagggcaccgacctgatcagattcgagcggaacatcgtgtgcaccagcatgaagcccatcaacgaggacctggacgagggcatcatggtggtgtacaagagaaacatcgtggcccacaccttcaaagtgcgggtgtaccagaaggtgctgaccttccggcggagctacgcctacatccacaccacatacctgctgggcagcaacaccgagtacgtggcccctcccatgtgggagatccaccacatcaacagccacagccagtgctacagcagctacagccgcgtgatcgccggcacagtgttcgtggcctaccaccgggacagctacgagaacaagaccatgcagctgatgcccgacgactacagcaacacccacagcaccagatacgtgaccgtgaaggaccagtggcacagcagaggcagcacctggctgtaccgggagacatgcaacctgaactgcatggtcaccatcaccaccgccagaagcaagtacccttaccacttcttcgccacctccaccggcgacgtggtggacatcagccccttctacaacggcaccaaccggaacgccagctacttcggcgagaacgccgacaagttcttcatcttccccaactacaccatcgtgtccgacttcggcagacccaacagcgctctggaaacccacagactggtggcctttctggaacgggccgacagcgtgatcagctgggacatccaggacgagaagaacgtgacctgccagctgaccttctgggaggcctctgagagaaccatcagaagcgaggccgaggacagctaccacttcagcagcgccaagatgaccgccaccttcctgagcaagaaacaggaagtgaacatgagcgactccgccctggactgcgtgagggacgaggccatcaacaagctgcagcagatcttcaacaccagctacaaccagacctacgagaagtatggcaatgtgtccgtgttcgagacaacaggcggcctggtggtgttctggcagggcatcaagcagaaaagcctggtggagctggaacggctcgccaaccggtccagcctgaacctgacccacaaccggaccaagcggagcaccgacggcaacaacgcaacccacctgtccaacatggaaagcgtgcacaacctggtgtacgcacagctgcagttcacctacgacaccctgcggggctacatcaacagagccctggcccagatcgccgaggcttggtgcgtggaccagcggcggaccctggaagtgttcaaagagctgtccaagatcaaccccagcgccatcctgagcgccatctacaacaagcctatcgccgccagattcatgggcgacgtgctgggcctggccagctgcgtgaccatcaaccagaccagcgtgaaggtgctgcgggacatgaacgtgaaagagagcccaggccgctgctactccagacccgtggtcatcttcaacttcgccaacagctcctacgtgcagtacggccagctgggcgaggacaacgagatcctgctggggaaccaccggaccgaggaatgccagctgcccagcctgaagatctttatcgccggcaacagcgcctacgagtatgtggactacctgttcaagcggatgatcgacctgagcagcatctccaccgtggacagcatgatcgccctggacatcgaccccctggaaaacaccgacttccgggtgctggaactgtacagccagaaagagctgcggagcagcaacgtgttcgacctggaagagatcatgcgggagttcaacagctacaagcagtgataa - 2082 Cmv gB sol 692 (SEQ ID NO: 6):MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNETIYNTTLKYGDVVGVNTIKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTTVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVIRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQ--CMV gH FL (SEQ ID NO: 7): 1-atgaggcctggcctgccctcctacctgatcatcctggccgtgtgcctgttcagccacctgctgtccagcagatacggcgccgaggccgtgagcgagcccctggacaaggctttccacctgctgctgaacacctacggcagacccatccggtttctgcgggagaacaccacccagtgcacctacaacagcagcctgcggaacagcaccgtcgtgagagagaacgccatcagcttcaactttttccagagctacaaccagtactacgtgttccacatgcccagatgcctgtttgccggccctctggccgagcagttcctgaaccaggtggacctgaccgagacactggaaagataccagcagcggctgaatacctacgccctggtgtccaaggacctggccagctaccggtcctttagccagcagctcaaggctcaggatagcctcggcgagcagcctaccaccgtgccccctcccatcgacctgagcatcccccacgtgtggatgcctccccagaccacccctcacggctggaccgagagccacaccacctccggcctgcacagaccccacttcaaccagacctgcatcctgttcgacggccacgacctgctgtttagcaccgtgaccccctgcctgcaccagggcttctacctgatcgacgagctgagatacgtgaagatcaccctgaccgaggatttcttcgtggtcaccgtgtccatcgacgacgacacccccatgctgctgatcttcggccacctgcccagagtgctgttcaaggccccctaccagcgggacaacttcatcctgcggcagaccgagaagcacgagctgctggtgctggtcaagaaggaccagctgaaccggcactcctacctgaaggaccccgacttcctggacgccgccctggacttcaactacctggacctgagcgccctgctgagaaacagcttccacagatacgccgtggacgtgctgaagtccggacggtgccagatgctcgatcggcggaccgtggagatggccttcgcctatgccctcgccctgttcgccgctgccagacaggaagaggctggcgcccaggtgtcagtgcccagagccctggatagacaggccgccctgctgcagatccaggaattcatgatcacctgcctgagccagaccccccctagaaccaccctgctgctgtaccccacagccgtggatctggccaagagggccctgtggacccccaaccagatcaccgacatcacaagcctcgtgcggctcgtgtacatcctgagcaagcagaaccagcagcacctgatcccccagtgggccctgagacagatcgccgacttcgccctgaagctgcacaagacccatctggccagctttctgagcgccttcgccaggcaggaactgtacctgatgggcagcctggtccacagcatgctggtgcataccaccgagcggcgggagatcttcatcgtggagacaggcctgtgtagcctggccgagctgtcccactttacccagctgctggcccaccctcaccacgagtacctgagcgacctgtacaccccctgcagcagcagcggcagacgggaccacagcctggaacggctgaccagactgttccccgatgccaccgtgcctgctacagtgcctgccgccctgtccatcctgtccaccatgcagcccagcaccctggaaaccttccccgacctgttctgcctgcccctgggcgagagctttagcgccctgaccgtgtccgagcacgtgtcctacatcgtgaccaatcagtacctgatcaagggcatcagctaccccgtgtccaccacagtcgtgggccagagcctgatcatcacccagaccgacagccagaccaagtgcgagctgacccggaacatgcacaccacacacagcatcaccgtggccctgaacatcagcctggaaaactgcgctttctgtcagtctgccctgctggaatacgacgatacccagggcgtgatcaacatcatgtacatgcacgacagcgacgacgtgctgttcgccctggacccctacaacgaggtggtggtgtccagcccccggacccactacctgatgctgctgaagaacggcaccgtgctggaagtgaccgacgtggtggtggacgccaccgacagcagactgctgatgatgagcgtgtacgccctgagcgccatcatcggcatctacctgctgtaccggatgctgaaaacctgctgataa -2232 Cmv gH FL (SEQ ID NO: 8);MRPGLPSYLIILAVCLFSHLLSSRYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQCTYNSSLRNSTVVRENAISFNFFQSYNQYYVFHMPRCLFAGPLAEQFLNQVDLTETLERYQQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTPHGWTESHTTSGLHRPHFNQTCILFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVVTVSIDDDIPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALALFAAARQEEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRAIWTPNQTTDTISLVRLVYILSKQNQQHLIPQWALRQTADFALKLHKTHLASFLSAFARQELYLMGSINHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHSLERLTRLFPDATVPATVPAALSILSTMQPSILETFPDLFCLPLGESFSALTVSEHVSYIVTNQYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITVALNISLENCAFCQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTDVVVDATDSRLLIINSVYALSAIIGIYLLYRMLKTC--CMV gH sol (SEQ ID NO: 9): 1-atgaggcctggcctgccctcctacctgatcatcctggccgtgtgcctgttcagccacctgctgtccagcagatacggcgccgaggccgtgagcgagcccctggacaaggctttccacctgctgctgaacacctacggcagacccatccggtttctgcgggagaacaccacccagtgcacctacaacagcagcctgcggaacagcaccgtcgtgagagagaacgccatcagcttcaactttttccagagctacaaccagtactacgtgttccacatgcccagatgcctgtttgccggccctctggccgagcagttcctgaaccaggtggacctgaccgagacactggaaagataccagcagcggctgaatacctacgccctggtgtccaaggacctggccagctaccggtcctttagccagcagctcaaggctcaggatagcctcggcgagcagcctaccaccgtgccccctcccatcgacctgagcatcccccacgtgtggatgcctccccagaccacccctcacggctggaccgagagccacaccacctccggcctgcacagaccccacttcaaccagacctgcatcctgttcgacggccacgacctgctgtttagcaccgtgaccccctgcctgcaccagggcttctacctgatcgacgagctgagatacgtgaagatcaccctgaccgaggatttcttcgtggtcaccgtgtccatcgacgacgacacccccatgctgctgatcttcggccacctgcccagagtgctgttcaaggccccctaccagcgggacaacttcatcctgcggcagaccgagaagcacgagctgctggtgctggtcaagaaggaccagctgaaccggcactcctacctgaaggaccccgacttcctggacgccgccctggacttcaactacctggacctgagcgccctgctgagaaacagcttccacagatacgccgtggacgtgctgaagtccggacggtgccagatgctcgatcggcggaccgtggagatggccttcgcctatgccctcgccctgttcgccgctgccagacaggaagaggctggcgcccaggtgtcagtgcccagagccctggatagacaggccgccctgctgcagatccaggaattcatgatcacctgcctgagccagaccccccctagaaccaccctgctgctgtaccccacagccgtggatctggccaagagggccctgtggacccccaaccagatcaccgacatcacaagcctcgtgcggctcgtgtacatcctgagcaagcagaaccagcagcacctgatcccccagtgggccctgagacagatcgccgacttcgccctgaagctgcacaagacccatctggccagctttctgagcgccttcgccaggcaggaactgtacctgatgggcagcctggtccacagcatgctggtgcataccaccgagcggcgggagatcttcatcgtggagacaggcctgtgtagcctggccgagctgtcccactttacccagctgctggcccaccctcaccacgagtacctgagcgacctgtacaccccctgcagcagcagcggcagacgggaccacagcctggaacggctgaccagactgttccccgatgccaccgtgcctgctacagtgcctgccgccctgtccatcctgtccaccatgcagcccagcaccctggaaaccttccccgacctgttctgcctgcccctgggcgagagctttagcgccctgaccgtgtccgagcacgtgtcctacatcgtgaccaatcagtacctgatcaagggcatcagctaccccgtgtccaccacagtcgtgggccagagcctgatcatcacccagaccgacagccagaccaagtgcgagctgaccaggaacatgcacaccacacacagcatcaccgtggccctgaacatcagcctggaaaactgcgctttctgtcagtctgccctgctggaatacgacgatacccagggcgtgatcaacatcatgtacatgcacgacagcgacgacgtgctgttcgccctggacccctacaacgaggtggtggtgtccagcccccggacccactacctgatgctgctgaagaacggcaccgtgctggaagtgaccgacgtggtggtggacgccaccgactgataa -2151 CMV gH sol (SEQ ID NO: 10):MRPGLPSYLIILAVCLFSHLLSSRYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQCTYNSSLRNSTVVRENAISFNFFQSYNQYYVFHMPRCLFAGPLAEQFLNQVDLTETLERYQQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTPHGWTESHTTSGLHRPHFNQICILFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVVIVSIDDDIPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRIVEMAFAYALALFAAARQEEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQITDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQELYLMGSLVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHSLERLTRLFPDATVPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYIVINQYLIKGISYPVSTIVVGQSLIITQTDSQTKCELTRNMHTTHSITVALNISLENCAFCQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTDVVVDATD-- CMV gL fl (SEQ ID NO: 11: 1-atgtgcagaaggcccgactgcggcttcagcttcagccctggacccgtgatcctgctgtggtgctgcctgctgctgcctatcgtgtcctctgccgccgtgtctgtggcccctacagccgccgagaaggtgccagccgagtgccccgagctgaccagaagatgcctgctgggcgaggtgttcgagggcgacaagtacgagagctggctgcggcccctggtcaacgtgaccggcagagatggccccctgagccagctgatccggtacagacccgtgacccccgaggccgccaatagcgtgctgctggacgaggccttcctggataccctggccctgctgtacaacaaccccgaccagctgagagccctgctgaccctgctgtccagcgacaccgcccccagatggatgaccgtgatgcggggctacagcgagtgtggagatggcagccctgccgtgtacacctgcgtggacgacctgtgcagaggctacgacctgaccagactgagctacggccggtccatcttcacagagcacgtgctgggcttcgagctggtgccccccagcctgttcaacgtggtggtggccatccggaacgaggccaccagaaccaacagagccgtgcggctgcctgtgtctacagccgctgcacctgagggcatcacactgttctacggcctgtacaacgccgtgaaagagttctgcctccggcaccagctggatccccccctgctgagacacctggacaagtactacgccggcctgcccccagagctgaagcagaccagagtgaacctgcccgcccacagcagatatggccctcaggccgtggacgccagatgataa - 840CMV gL FL (SEQ ID NO: 12):MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEHVLGFELVPPSLFNVVVATRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR--CMV gM FL (SEQ ID NO: 13): 1-atggcccccagccacgtggacaaagtgaacaccaggacttggagcgccagcatcgtgttcatggtgctgaccttcgtgaacgtgtccgtgcacctggtgctgtccaacttcccccacctgggctacccctgcgtgtactaccacgtggtggacttcgagcggctgaacatgagcgcctacaacgtgatgcacctgcacacccccatgctgtttctggacagcgtgcagctcgtgtgctacgccgtgttcatgcagctggtgtttctggccgtgaccatctactacctcgtgtgctggatcaagatcagcatgcggaaggacaagggcatgagcctgaaccagagcacccgggacatcagctacatgggcgacagcctgaccgccttcctgttcatcctgagcatggacaccttccagctgttcaccctgaccatgagcttccggctgcccagcatgatcgccttcatggccgccgtgcactttttctgtctgaccatcttcaacgtgtccatggtcacccagtaccggtcctacaagcggagcctgttcttcttctcccggctgcaccccaagctgaagggcaccgtgcagttccggaccctgatcgtgaacctggtggaggtggccctgggcttcaataccaccgtggtggctatggccctgtgctacggcttcggcaacaacttcttcgtgcggaccggccatatggtgctggccgtgttcgtggtgtacgccatcatcagcatcatctactttctgctgatcgaggccgtgttcttccagtacgtgaaggtgcagttcggctaccatctgggcgcctttttcggcctgtgcggcctgatctaccccatcgtgcagtacgacaccttcctgagcaacgagtaccggaccggcatcagctggtccttcggaatgctgttcttcatctgggccatgttcaccacctgcagagccgtgcggtacttcagaggcagaggcagcggctccgtgaagtaccaggccctggccacagcctctggcgaagaggtggccgccctgagccaccacgacagcctggaaagcagacggctgcgggaggaagaggacgacgacgacgaggacttcgaggacgcctgataa - 1119CMV gM FL (SEQ ID NO: 14):MAPSHVDKVNTRTWSASIVFMVLIFVNVSVHLVLSNFPHLGYPCVYYHVVDFERLNMSAYNVMHLHIPMLFLDSVQLVCYAVFMQLVFLAVTIYYLVCWIKISMRKDKGMSLNQSTRDISYMGDSLTAFLFILSMDTFQLFTLIMSFRLPSMIAFMAAVHFFCLTIFNVSMVTQYRSYKRSLFFFSRLHPKLKGTVQFRTLIVNLVEVALGFNITVVAMALCYGFGNNFFVRTGHMVLAVFVVYAIISITYFLLIEAVFFQYVKVQFGYHLGAFFGLCGLIYPIVQYDTFLSNEYRTGISWSEGMLFFIWAMETTCRAVRYFRGRGSGSVKYQALATASGEEVAALSHHDSLESRRLREEEDDDDEDFEDA--CMV gN FL (SEQ ID NO: 15): 1-atggaatggaacaccctggtcctgggcctgctggtgctgtctgtcgtggccagcagcaacaacacatccacagccagcacccctagacctagcagcagcacccacgccagcactaccgtgaaggctaccaccgtggccaccacaagcaccaccactgctaccagcaccagctccaccacctctgccaagcctggctctaccacacacgaccccaacgtgatgaggccccacgcccacaacgacttctacaacgctcactgcaccagccacatgtacgagctgtccctgagcagctttgccgcctggtggaccatgctgaacgccctgatcctgatgggcgccttctgcatcgtgctgcggcactgctgcttccagaacttcaccgccaccaccaccaagggctactgataa - 411 CMV gN FL (SEQ ID NO: 16):MEWNTLVLGLLVLSVVASSNNTSTASTPRPSSSTHASTTVKATTVATTSTTTATSISSTTSAKPGSTTHDPNVMRPHAHNDFYNAHCTSHMYELSLSSFAAWWTMLNALILMGAFCTVLRHCCFQNFTATTTKGY--CMV gO FL (SEQ ID NO: 17): 1-atgggcaagaaagaaatgatcatggtcaagggcatccccaagatcatgctgctgattagcatcacctttctgctgctgtccctgatcaactgcaacgtgctggtcaacagccggggcaccagaagatcctggccctacaccgtgctgtcctaccggggcaaagagatcctgaagaagcagaaagaggacatcctgaagcggctgatgagcaccagcagcgacggctaccggttcctgatgtaccccagccagcagaaattccacgccatcgtgatcagcatggacaagttcccccaggactacatcctggccggacccatccggaacgacagcatcacccacatgtggttcgacttctacagcacccagctgcggaagcccgccaaatacgtgtacagcgagtacaaccacaccgcccacaagatcaccctgaggcctcccccttgtggcaccgtgcccagcatgaactgcctgagcgagatgctgaacgtgtccaagcggaacgacaccggcgagaagggctgcggcaacttcaccaccttcaaccccatgttcttcaacgtgccccggtggaacaccaagctgtacatcggcagcaacaaagtgaacgtggacagccagaccatctactttctgggcctgaccgccctgctgctgagatacgcccagcggaactgcacccggtccttctacctggtcaacgccatgagccggaacctgttccgggtgcccaagtacatcaacggcaccaagctgaagaacaccatgcggaagctgaagcggaagcaggccctggtcaaagagcagccccagaagaagaacaagaagtcccagagcaccaccaccccctacctgagctacaccacctccaccgccttcaacgtgaccaccaacgtgacctacagcgccacagccgccgtgaccagagtggccacaagcaccaccggctaccggcccgacagcaactttatgaagtccatcatggccacccagctgagagatctggccacctgggtgtacaccaccctgcggtacagaaacgagcccttctgcaagcccgaccggaacagaaccgccgtgagcgagttcatgaagaatacccacgtgctgatcagaaacgagacaccctacaccatctacggcaccctggacatgagcagcctgtactacaacgagacaatgagcgtggagaacgagacagccagcgacaacaacgaaaccacccccacctcccccagcacccggttccagcggaccttcatcgaccccctgtgggactacctggacagcctgctgttcctggacaagatccggaacttcagcctgcagctgcccgcctacggcaatctgaccccccctgagcacagaagggccgccaacctgagcaccctgaacagcctgtggtggtggagccagtgataa - 1422 CMV gO FL (SEQ ID NO: 18):MGKKEMIMVKGIPKIMLLISITFLLLSLINCNVLVNSRGIRRSWPYTVLSYRGKEILKKQKEDILKRLMSTSSDGYRFLMYPSQQKFHAIVISMDKFPQDYILAGPIRNDSITHMWFDFYSTQLRKPAKYVYSEYNHTAHKITLRPPPCGTVPSMNCLSEMLNVSKRNDTGEKGCGNFTTFNPMFFNVPRWNTKLYIGSNKVNVDSQTIYFLGLTALLLRYAQRNCTRSFYLVNAMSRNLFRVPKYINGTKLKNIMRKLKRKQALVKEQPQKKNKKSQSITTPYLSYTTSTAFNVTINVTYSATAAVIRVATSTTGYRPDSNFMKSIMATQLRDLATWVYTTLRYRNEPFCKPDRNRTAVSEFMKNTHVLIRNETPYTIYGILDMSSLYYNETMSVENETASDNNETTPTSPSTRFQRTFIDPLWDYLDSLLFLDKIRNFSLQLPAYGNLIPPEHRRAANLSTLNSLSQ-- CMV UL128 FL (SEQ ID NO: 19): 1-atgagccccaaggacctgacccccttcctgacaaccctgtggctgctcctgggccatagcagagtgcctagagtgcgggccgaggaatgctgcgagttcatcaacgtgaaccacccccccgagcggtgctacgacttcaagatgtgcaaccggttcaccgtggccctgagatgccccgacggcgaagtgtgctacagccccgagaaaaccgccgagatccggggcatcgtgaccaccatgacccacagcctgacccggcaggtggtgcacaacaagctgaccagctgcaactacaaccccctgtacctggaagccgacggccggatcagatgcggcaaagtgaacgacaaggcccagtacctgctgggagccgccggaagcgtgccctaccggtggatcaacctggaatacgacaagatcacccggatcgtgggcctggaccagtacctggaaagcgtgaagaagcacaagcggctggacgtgtgcagagccaagatgggctacatgctgcagtgataa - 519CMV UL128 FL (SEQ ID NO: 20):MSPKDLTPFLTTLWLLLGHSRVPRVRAEECCEFINVNHPPERCYDFKMCNRFTVALRCPDGEVCYSPEKTAEIRGIVTTMTHSLTRQVVHNKLISCNYNPLYLEADGRIRCGKVNDKAQYLLGAAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAKMGYMLQ-- CMV UL130 FL (SEQ ID NO: 21): 1-atgctgcggctgctgctgagacaccacttccactgcctgctgctgtgtgccgtgtgggccaccccttgtctggccagcccttggagcaccctgaccgccaaccagaaccctagccccccttggtccaagctgacctacagcaagccccacgacgccgccaccttctactgcccctttctgtaccccagccctcccagaagccccctgcagttcagcggcttccagagagtgtccaccggccctgagtgccggaacgagacactgtacctgctgtacaaccgggagggccagacactggtggagcggagcagcacctgggtgaaaaaagtgatctggtatctgagcggccggaaccagaccatcctgcagcggatgcccagaaccgccagcaagcccagcgacggcaacgtgcagatcagcgtggaggacgccaaaatcttcggcgcccacatggtgcccaagcagaccaagctgctgagattcgtggtcaacgacggcaccagatatcagatgtgcgtgatgaagctggaaagctgggcccacgtgttccgggactactccgtgagcttccaggtccggctgaccttcaccgaggccaacaaccagacctacaccttctgcacccaccccaacctgatcgtgtgataa - 648 CMV UL130 FL (SEQ ID NO: 22):MLRLLLRHHFHCLLLCAVWATPCLASPWSTLTANQNPSPPWSKLTYSKPHDAATFYCPFLYPSPPRSPLQFSGFQRVSTGPECRNETLYLLYNREGQTLVERSSTWVKKVIWYLSGRNQTILQRMPRTASKPSDGNVQISVEDAKIFGAHMVPKQTKLLRFVVNDGTRYQMCVMKLESWAHVFRDYSVSFQVRLIFTEANNQTYTFCTHPNLIV-- CMV UL131 FL (SEQ ID NO: 23): 1-atgcggctgtgcagagtgtggctgtccgtgtgcctgtgtgccgtggtgctgggccagtgccagagagagacagccgagaagaacgactactaccgggtgccccactactgggatgcctgcagcagagccctgcccgaccagacccggtacaaatacgtggagcagctcgtggacctgaccctgaactaccactacgacgccagccacggcctggacaacttcgacgtgctgaagcggatcaacgtgaccgaggtgtccctgctgatcagcgacttccggcggcagaacagaagaggcggcaccaacaagcggaccaccttcaacgccgctggctctctggcccctcacgccagatccctggaattcagcgtgcggctgttcgccaactgataa - 393 CMV UL131 FL (SEQ ID NO: 24):MRLCRVWLSVCLCAVVLGQCQRETAEKNDYYRVPHYWDACSRALPDQTRYKYVEQLVDLTLNYHYDASHGLDNFDVLKRINVTEVSLLISDFRRQNRRGGTNKRTTFNAAGSLAPHARSLEFSVRLFAN--

What is claimed:
 1. A method for treating or preventing an infectiousdisease comprising: (i) administering to a subject in need thereof atleast once a therapeutically effective amount of a priming compositioncomprising a self-replicating RNA molecule that encodes a firstpolypeptide antigen that comprises a first epitope from a pathogen; and(ii) subsequently administering the subject at least once atherapeutically effective amount of a boosting composition comprising asecond polypeptide antigen that comprises a second epitope from saidpathogen; wherein said first epitope and second epitope are the sameepitope.
 2. The method of claim 1, wherein said first polypeptideantigen and second polypeptide antigen are substantially the same. 3.The method of claim 1, wherein said first polypeptide antigen is asoluble or membrane anchored polypeptide, and said second polypeptideantigen is a soluble polypeptide.
 4. The method of claim 1, wherein saidfirst polypeptide antigen is a fusion polypeptide.
 5. The method ofclaim 1, wherein said second polypeptide antigen is a fusionpolypeptide.
 6. The method of claim 1, wherein the self-replicating RNAis an alphavirus-derived RNA replicon.